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Chronic histiocytic intervillositis of unknown origin (CHI) is a rare placental disorder associated with adverse pregnancy outcomes, frequent recurrence, and a lack of effective preventive strategies. Recent insights indicate a potential link between CHI-associated inflammatory lesions and the inflammasome pathway, suggesting innovative therapeutic avenues. Here we show a potential role of the inflammasome pathway in CHI through comprehensive transcriptomic analysis of grade 2 or 3 histopathologic CHI samples, paired with placental controls. Additionally, we present case studies of three individuals with recurrent CHI, who have undergone treatment with anakinra and colchicine throughout pregnancy, resulting in improved perinatal outcomes. Notably, all cases are characterized by the birth of healthy, full-term infants, with reduced or absent intervillositis recurrence. Placental assessment unveils heightened activation of the NLRP3-PYCARD inflammasome pathway and IL-1β processing in CHI samples, with downregulation observed in treated pregnancy samples, devoid of intervillositis. Collectively, these findings suggest a potential therapeutic role for targeting the inflammasome pathway in preventing recurrent CHI in pregnant individuals., (© 2024. The Author(s).)

Background: Recurrent spontaneous abortion (RSA) is defined as two or more consecutive spontaneous abortions before 20 weeks with the same spouse [1]. However, approximately 50% of RSA cases of unknown cause are classified as unexplained recurrent spontaneous abortion (URSA). Potential factors include decreased trophoblast cell migration and invasion, leading to impaired placental implantation and maintenance of the normal maternal-fetal interface. However, the mechanism of this pathogenesis remains unknown. In this study, we investigated the potential role and mechanism of KLF4 in regulating URSA by influencing the invasion and migration ability of trophoblast cells., Methods: We firstly identified 817 differentially expressed genes by performing a difference analysis of the dataset GSE121950 [2] related to recurrent abortion, and intersected the top 10 genes obtained respectively by the three algorithms: DMNC, MNC, and EPC using Venn Diagram.To detect the expression levels of core genes, villi samples were obtained from normal pregnant women and patients with URSA. RT-qPCR analysis revealed a significant difference in KLF4 mRNA expression and KLF4 was then analyzed. Trophoblast cell lines HTR8 and JEG3 were used to investigate the effect of KLF4 on trophoblastic function. Wound healing and transwell assays was performed to detect the invasion and migration of trophoblast cells. The expression of epithelial-mesenchymal transition(EMT) molecules were detected by RT-qPCR and western blot. Promoter detection and epigenetic modification were detected by chromatin immunoprecipitation (ChIP) assay. Molecular nuclear localization was detected by immunofluorescence and subcellular fractionation. Miscarried mice model was used to study the effects of KLF4 on URSA induced by reduced trophoblast invasion and migration., Results: KLF4 is highly expressed in the villi of patients with URSA. KLF4 inhibits the expression level of H3R2ME2a in trophoblast cells by regulating the transcriptional level and nuclear translocation of PRMT6, thereby inhibiting the possible regulatory mechanism of trophoblastic invasion and providing a potential treatment strategy for URSA in vivo., Conclusions: The KLF4/PRMT6/H3R2ME2a axis regulates mechanisms associated with unexplained recurrent spontaneous abortion by regulating trophoblast function., (© 2024. The Author(s).)

The placenta, a pivotal player in the prenatal environment, holds crucial insights into early developmental pathways and future health outcomes. In this study, we explored genetic molecular regulation in chorionic villus samples (CVS) from the first trimester and placenta tissue at birth. We assessed quantitative trait locus (QTL) mapping on DNA methylation and gene expression data in a Finnish cohort of 574 individuals. We found more QTLs in birth placenta than in first-trimester placenta. Nevertheless, a substantial amount of associations overlapped in their effects and showed consistent direction in both tissues, with increasing molecular genetic effects from early pregnancy to birth placenta. The identified QTLs in birth placenta were most enriched in genes with placenta-specific expression. Conducting a phenome-wide-association study (PheWAS) on the associated SNPs, we observed numerous overlaps with genome-wide association study (GWAS) hits (spanning 57 distinct traits and 23 SNPs), with notable enrichments for immunological, skeletal, and respiratory traits. The QTL-SNP rs1737028 (chr6:29737993) presented with the highest number of GWAS hits. This SNP was related to HLA-G expression via DNA methylation and was associated with various immune, respiratory, and psychiatric traits. Our findings implicate increasing genetic molecular regulation during the course of pregnancy and support the involvement of placenta gene regulation, particularly in immunological traits. This study presents a framework for understanding placenta-specific gene regulation during pregnancy and its connection to health-related traits., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Gestational diabetes mellitus (GDM) is a known risk factor for stillbirth (Rosenstein et al., 2012) [1]. Delayed villous maturation (DVM), predominantly seen in term placentas in pregnancies complicated with glucose dysmetabolism, may in part be a consequence of excessive maternal glucose leading to release of fetal insulin and other growth factors that promote excessive placental growth at the expense of villous maturation (Redline, 2012) [2]., Cases: We present three cases of under-diagnosed/treated glucose dysmetabolism in women in their first pregnancies cared for in other hospitals in the United Kingdom (UK) with the fatal fetal/neonatal outcomes and confirmed DVM in the placenta and congenital pneumonia on post-mortem examination in all three cases., Conclusion: This cluster supports a hypothesis that DVM and glucose dysmentabolism may make babies more susceptible to severe perinatal infection. All three cases received the antenatal care in their subsequent pregnancies in our unit and had confirmed glucose dysmetabolism which was treated and resulted in healthy babies., Competing Interests: Declaration of competing interest None declared., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Mesenchymal cells within theplacental villi play a crucial role in shaping the morphology of branching structures and driving the development of blood vessels. However, the markers and functions of placental villous pericytes (PVPs) as distinct subgroups of placental villous mesenchymal cells, remain unclear. Therefore, in this study, the markers and functions of PVPs were investigated. Single-cell sequencing data from the first-trimester placental villi was obtained and the Seurat tool was used to identify PVP markers. Gene Ontology (GO) analysis of specific genes was performed using the DAVID database. The Cellchat tool was employed to investigate the interaction signals between the PVPs and other cells. Expression of the PVP markers was confirmed using immunofluorescence. Presence of extracellular vesicles in the placental villous mesenchyme and PVPs was examined by transmission electron microscopy. Our findings revealed that renin (REN) and amphiregulin (AREG)-positive fibroblasts in the placental villi specifically expressed several classic pericyte markers. In the first trimester, certain conserved functions of pericytes were observed and they displayed tissue-specific functions such as in the integrin-mediated signaling pathway and extracellular exosomes. Moreover, the placental villous mesenchyme was found to be rich in extracellular vesicles. AREG is specifically transcribed in the first trimester PVPs, however, its protein was located in syncytiotrophoblasts. These insights contribute to a comprehensive understanding of early placental development and offer new therapeutic targets for placenta-derived pregnancy complications., Competing Interests: Declaration of Competing Interest The author declares that he has no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

Chronic histiocytic intervillositis (CHI) is a recurrent placental lesion where maternal macrophages infiltrate the intervillous space. Its cause is unknown, though due to similarities to rejected allografts one hypothesis is that CHI represents maternal-fetal rejection. Here, virtual crossmatching was applied to healthy pregnancies and those with a history of CHI. Anti-HLA antibodies, measured by Luminex, were present in slightly more controls than CHI (8/17 (47.1%) vs 5/14 (35.7%)), but there was no significant difference in levels of sensitisation or fetal specific antibodies. Quantification of immunohistochemical staining for HLA-Class II was increased in syncytiotrophoblast of placentas with CHI (Grade 0.44 [IQR 0.1-0.7]) compared to healthy controls (0.06 [IQR 0-0.2]) and subsequent pregnancies (0.13 [IQR 0-0.3]) (P = 0.0004). HLA-Class II expression was positively related both to the severity of CHI (r = 0.67) and C4d deposition (r = 0.48). There was no difference in overall C4d and HLA-Class I immunostaining. Though increased anti-HLA antibodies were not evident in CHI, increased expression of HLA-Class II at the maternal-fetal interface suggests that they may be relevant in its pathogenesis. Further investigation of antibodies immediately after diagnosis is warranted in a larger cohort of CHI cases to better understand the role of HLA in its pathophysiology., (© 2024. The Author(s).)

Preeclampsia is a disorder of pregnancy characterized by endothelial dysfunction, abnormal placentation, systemic inflammation, and altered immune reaction. The aim of this study was to investigate the immune checkpoint molecules TIM-3 and Gal-9 on macrophages and Hofbauer cells (HBC) in the placenta of preeclampsia patients. Immunohistochemistry and Immunofluorescence was used to characterize the expression of the macrophage markers CD68 and CD163, CK7 and the proteins TIM-3 and Gal-9 in the placentas of preeclampsia patients comparing it to the placentas of healthy pregnancies. Double immunofluorescence staining (TIM-3 with CD3/CD19/CD56) was used to analyze the TIM-3 expression on other immune cells (T cells, B cells, NK cells) within the chorionic villi. The expression of TIM-3 on decidual macrophages did not significantly differ between the preeclamptic and the control group (p = 0.487). When looking at the different offspring we saw an upregulation of TIM-3 expression on decidual macrophages in preeclamptic placentas with female offspring (p = 0.049). On Hofbauer cells within the chorionic villi, the TIM-3 expression was significantly downregulated in preeclamptic cases without a sex-specific difference (p < 0.001). Looking at the protein Gal-9 the expression was proven to be downregulated both, on decidual macrophages (p = 0.003) and on Hofbauer cells (p = 0.002) within preeclamptic placentas compared to healthy controls. This was only significant in male offspring (p < 0.001 and p = 0.013) but not in female offspring (p = 0.360 and p = 0.068). While TIM-3 expression within the extravillious trophoblast and the syncytiotrophoblast was significantly downregulated (p < 0.001 and p = 0.012) in preeclampsia, the expression of Gal-9 was upregulated in (p < 0.001 and p < 0.001) compared to healthy controls. The local variations of the immune checkpoint molecules TIM-3 and Gal-9 in the placenta may contribute to the inflammation observed in preeclamptic patients. It could therefore contribute to the pathogenesis and be an important target in the treatment of preeclampsia., Competing Interests: Declaration of Competing Interest N.D. reports funding from MSD, Novartis, Pfizer, Roche, AstraZeneca, TEVA, Mentor, and MCI Healthcare. U.J. received travel money from pfm. C.D. is funded by Roche, AstraZeneca, TEVA, Mentor, and MCI Healthcare. All other authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The environment created during embryogenesis contributes to reducing aberrations that drive structural malformations and tumorigenesis. In this study, we investigate the anti-cancer effect of mesenchymal stem cells (MSCs) derived from 2 different gestational tissues, the amniotic fluid (AF) and the chorionic villi (CV), with emphasis on their secretome. Transcriptomic analysis was performed on patient-derived AF- and CV-MSCs collected during prenatal diagnosis and identified both mRNAs and lncRNAs, involved in tissue homeostasis and inhibiting biological processes associated with the etiology of aggressive cancers while regulating immune pathways shown to be important in chronic disorders. Secretome enrichment analysis also identified soluble moieties involved in target cell regulation, tissue homeostasis, and cancer cell inhibition through the highlighted Wnt, TNF, and TGF-β signaling pathways. Transcriptomic data were experimentally confirmed through in vitro assays, by evaluating the anti-cancer effect of the media conditioned by AF- and CV-MSCs and the exosomes derived from them on ovarian cancer cells, revealing inhibitory effects in 2D (by reducing cell viability and inducing apoptosis) and in 3D conditions (by negatively interfering with spheroid formation). These data provide molecular insights into the potential role of gestational tissues-derived MSCs as source of anti-cancer factors, paving the way for the development of therapeutics to create a pro-regenerative environment for tissue restoration following injury, disease, or against degenerative disorders., (© The Author(s) 2024. Published by Oxford University Press.)

Objective: This study aimed to explore the specific pathways by which HOX transcript antisense intergenic RNA contributes to the pathogenesis of unexplained recurrent spontaneous abortion., Methods: Real-time quantitative PCR was employed to assess the differential expression levels of HOX transcript antisense intergenic RNA in chorionic villi tissues from unexplained recurrent spontaneous abortion patients and women with voluntarily terminated pregnancies. HTR-8/SVneo served as a cellular model. Knockdown and overexpression of HOX transcript antisense intergenic RNA in the cells were achieved through siRNA transfection and pcDNA3.1 transfection, respectively. Cell viability, migration, and invasion were evaluated using cell counting kit-8, scratch, and Transwell assays, respectively. The interaction among the HOX transcript antisense intergenic RNA /miR-1277-5p/fibrillin 2 axis was predicted through bioinformatics analysis and confirmed through in vitro experiments. Furthermore, the regulatory effects of the HOX transcript antisense intergenic RNA /miR-1277-5p/fibrillin 2 signaling axis on cellular behaviors were validated in HTR-8/SVneo cells., Results: We found that HOX transcript antisense intergenic RNA was downregulated in chorionic villi tissues from unexplained recurrent spontaneous abortion patients. Overexpression of HOX transcript antisense intergenic RNA significantly enhanced the viability, migration, and invasion of HTR-8/SVneo cells, while knockdown of HOX transcript antisense intergenic RNA had the opposite effects. We further confirmed the regulatory effect of the HOX transcript antisense intergenic RNA /miR-1277-5p/fibrillin 2 signaling axis in unexplained recurrent spontaneous abortion. Specifically, HOX transcript antisense intergenic RNA and fibrillin 2 were found to reduce the risk of unexplained recurrent spontaneous abortion by enhancing cell viability, migration, and invasion, whereas miR-1277-5p exerted the opposite effects., Conclusion: HOX transcript antisense intergenic RNA promotes unexplained recurrent spontaneous abortion development by targeting inhibition of miR-1277-5p/fibrillin 2 axis., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The migration, proliferation, and apoptosis of trophoblastic cells play a crucial role in ensuring the effective preservation of pregnancy at the maternal-fetal interface. Any deviations in the structure and function of these cells might potentially result in the development of numerous pregnancy-related disorders, including missed abortion (MA). This study involved the examination of semaphorin 4A ( SEMA4A ) expression in missed abortion (n = 18) and normal early pregnancy ( n = 18) villus. The findings of this study indicate a statistically significant decrease in the expression of SEMA4A in the villi of individuals diagnosed with missed abortion, as compared to the control group. The results of our vitro study showed that SEMA4A promoted the migration and proliferation of trophoblast cells and inhibited their apoptosis. Subsequent studies have shown that SEMA4A may be involved in regulating p-STAT3/STAT3 , MMP9 , bcl-2 , and BAX levels. In summary, the findings of this study indicate a correlation between the decreased level of SEMA4A in chorionic villi and missed abortion. These results offer novel theoretical insights into the proper implantation and development of SEMA4A embryos at the maternal-fetal interface.

A comparative study was carried out to analyze the number of syncytial knots and the intensity of expression of vascular endothelial growth factor (VEGF) in the villi of the monochorionic diamniotic placenta in pregnancies complicated by the syndrome of selective fetal growth restriction (sFGR). We performed a morphological analysis of 32 monochorionic diamniotic placentas after term delivery which were divided into two groups. The main group included the placentas of 15 puerperas whose pregnancies were complicated by sFGR. The control group included twin placentas of 17 puerperas without signs of sFGR. The number of syncytial knots was determined by histological studies, and the levels of VEGF expression in syncytiotrophoblast and capillary endotheliocytes of the placental villi were determined by immunohistochemical studies. The study showed an increase in the number of syncytial knots in the villi of the placental part of the fetus with sFGR which indicated the development of preplacental hypoxia. A significant increase in the level of VEGF expression in the syncytiotrophoblast and vascular endothelium of the villi should be considered as a manifestation of a compensatory adaptational response to hypoxia, though it is insufficient to prevent the development of sFGR., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

The early-stage placental barrier is characterized by a lack of fetal circulation and by a thick trophoblastic barrier, whereas the later-stage placenta consists of vascularized chorionic villi encased in a thin, differentiated trophoblast layer, ideal for nutrient transport. In this work, predictive models of early- and late-stage placental transport are created using blastocyst-derived placental stem cells (PSCs) by modulating PSC differentiation and model vascularization. PSC differentiation results in a thinner, fused trophoblast layer, as well as an increase in human chorionic gonadotropin secretion, barrier permeability, and secretion of certain inflammatory cytokines, which are consistent with in vivo findings. Further, gene expression confirms this shift toward a differentiated trophoblast subtype. Vascularization results in a molecule type- and size-dependent change in dextran and insulin permeability. These results demonstrate that trophoblast differentiation and vascularization have critical effects on placental barrier permeability and that this model can be used as a predictive measure to assess fetal toxicity of xenobiotic substances at different stages of pregnancy., (© 2023 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.)

Background: It has been long known that thyroid hormone regulates placental villi development, which is associated with the occurrence of miscarriage. However, whether abnormal thyroid hormone metabolism and transport in placental villi are involved in miscarriage is still to be verified., Methods: Placental villi of elective terminations of pregnancies (ETPs) and miscarriage were collected. Proliferative activity and apoptosis of villi trophoblasts and angiogenesis were detected by TUNEL and immunochemistry. The expressions of thyroid hormone receptors (THRs), transthyretin (TTR), monocarboxylate transporter 8 (MCT8), organic anion transporting polypeptides 1A1 (OATP1A1), deiodinase 2 (Dio2) and Dio3 were examined by RT-PCR, Western blot, immunohistochemistry and immunofluorescence. JEG3 cell was treated with iopanoic acid (IOP), an inhibitor of Dio2 activity, the expressions of Dio2, placenta growth factor (PLGF) and sFlt1 were detected by RT-PCR and Western blot., Results: Cell proliferation was suppressed and apoptosis was increased in placental villi cytotrophoblasts of miscarriage. CD34 + vessel number and vascular endothelial growth factor (VEGF) protein abundance were decreased in miscarriage. In miscarriage group, the gene expression of Dio2, Dio3, TTR and THRα, but not THRβ, MCT8 and OATP1A1, were downregulated. The protein abundances of TTR and THRα were downregulated in miscarriage group, but not THRβ. The protein abundance of Dio2 in miscarriage villi was decreased compared with that in ETP. In JEG3 cells, the gene expression of PLGF was decreased and the expression of sFlt1 was increased in IOP treatment; The protein abundance of Dio2 was downregulated but the gene expression of Dio2 was unaffected in IOP treatment., Conclusion: Thyroid hormone transport and metabolism in miscarriage were disturbed and may impaired angiogenesis of placental villi, which was associated with the occurrence of miscarriage., (© 2023. The Author(s).)

The interaction between trophoblasts, stroma cells, and immune cells at the maternal-fetal interface constitutes the functional units of the placenta, which is crucial for successful pregnancy outcomes. However, the investigation of this intricate interplay is restricted due to the absence of efficient experimental models. To address this challenge, a robust, reliable methodology for generating placenta villi organoids (PVOs) from early, late, or diseased pregnancies using air-liquid surface culture is developed. PVOs contain cytotrophoblasts that can self-renew and differentiate directly, along with stromal elements that retain native immune cells. Analysis of scRNA sequencing and WES data reveals that PVOs faithfully recapitulate the cellular components and genetic alterations of the corresponding source tissue. Additionally, PVOs derived from patients with preeclampsia exhibit specific pathological features such as inflammation, antiangiogenic imbalance, and decreased syncytin expression. The PVO-based propagation of primary placenta villi should enable a deeper investigation of placenta development and exploration of the underlying pathogenesis and therapeutics of placenta-originated diseases., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Introduction: Chronic histiocytic intervillositis (CHI) is a rare histopathological lesion in the placenta characterized by an infiltrate of CD68 + cells in the intervillous space. CHI is associated with adverse pregnancy outcomes such as miscarriage, fetal growth restriction, and (late) intrauterine fetal death. The adverse pregnancy outcomes and a variable recurrence rate of 25-100% underline its clinical relevance. The pathophysiologic mechanism of CHI is unclear, but it appears to be immunologically driven. The aim of this study was to obtain a better understanding of the phenotype of the cellular infiltrate in CHI., Method: We used imaging mass cytometry to achieve in-depth visualization of the intervillous maternal immune cells and investigated their spatial orientation in situ in relation to the fetal syncytiotrophoblast., Results: We found three phenotypically distinct CD68 + HLA-DR + CD38 + cell clusters that were unique for CHI. Additionally, syncytiotrophoblast cells in the vicinity of these CD68 + HLA-DR + CD38 + cells showed decreased expression of the immunosuppressive enzyme CD39., Discussion: The current results provide novel insight into the phenotype of CD68 + cells in CHI. The identification of unique CD68 + cell clusters will allow more detailed analysis of their function and could result in novel therapeutic targets for CHI., Competing Interests: Declaration of competing interest The authors do not have a conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Aim: To investigate the expression of autophagy mediated by the hypoxia-inducible factor 1α (HIF-1α)/BNIP3 signaling pathway in villus tissues of missed abortion and HTR-8/SVneo cells and to elucidate the association of HIF-1α and BNIP3 in autophagy of missed abortion., Methods: Villus tissues from 30 healthy women with induced abortion and 35 patients with missed abortion were collected, and HTR-8/SVneo cells were cultured under hypoxia and transfected with HIF-1α-siRNA. Real-time polymerase chain reaction was utilized to measure the mRNA levels of HIF-1α and BNIP3; Western blotting was performed to determine the protein levels of HIF-1α, BNIP3, LC3 II/I, and Beclin 1 in villus tissues and HTR-8/SVneo cells. Cellular invasion activity was detected by transwell matrigel assay. The level of autophagy was confirmed by transmission electron microscopy of autophagosome formation., Results: The mRNA levels of HIF-1α and BNIP3 were significantly lower in the missed abortion villi than in the induced abortion samples. The protein levels of HIF-1α, BNIP3, Beclin 1, and LC3II/I were significantly decreased in villus tissues from missed abortion, and autophagosomes were significantly decreased in villus tissues from missed abortion. Under hypoxia, the mRNA expression of HIF-1α and BNIP3 was inhibited after silencing HIF-1α by RNAi, while the protein expression of HIF-1α, BNIP3, Beclin1, and LC3II/I was significantly downregulated. The number of invading cells was significantly decreased, and autophagosomes were significantly decreased after silencing HIF-1α by RNAi in HTR-8/SVneo cells., Conclusions: Autophagy mediated by the HIF-1α/BNIP3 signaling pathway in villous trophoblast cells may be associated with the progression and development of missed abortion., (© 2023 Japan Society of Obstetrics and Gynecology.)

Background: Unexplained recurrent spontaneous abortion (URSA) is one of the most challenging conditions frustrates women of childbearing age profoundly. The gene expression patterns and biological characteristics of placental villus in patients with URSA remain largely unknown. The aim of our study was to identify potential lncRNAs as well as their action mechanisms in URSA., Method: The ceRNA microarray was used to identify the mRNA and lncRNA expression profiles of URSA patients and normal pregnancy. Functional enrichment analyses for differentially expressed mRNAs in URSA were performed. Protein-protein interaction analysis of differentially expressed mRNAs was performed to identify hub genes and key modules. Subsequently, the co-dysregulated ceRNA network of URSA was established, and the enrichment analyses for the mRNAs in the ceRNA network was implemented. qRT-PCR was performed to validated the expression of key ENST00000429019 and mRNAs in URSA., Results: We found that URSA placental villus have distinct mRNA and lncRNA expression profiles through ceRNA microarray, with a total of 347 mRNAs and 361 lncRNAs differentially expressed compared with controls. The functional enrichment analysis revealed that ncRNA processing, DNA replication, cell cycle, apoptosis, cytokine-mediated signaling pathway, ECM-receptor interaction were the potentially disrupted pathways in URSA patients. Then we constructed a co-dysregulated ceRNA network and found differentially expressed mRNAs were regulated by a small fraction of hub lncRNAs. Finally, we found a key network of ENST00000429019 and three cell proliferation or apoptosis related key mRNAs (CDCA3, KIFC1, NCAPH), and validated their expression and regulation in tissue and cellular levels., Conclusions: This study identified a key ceRNA network, which might take part in URSA and correlate with cell proliferation and apoptosis. Optimistically, this study may deepen our apprehensions about the underlying molecular and biological causes of URSA and provide an important theoretical basis for future therapeutic strategies for patients with URSA., (© 2023. The Author(s).)

Preeclampsia is a pregnancy-specific disease which is characterized by abnormal placentation, endothelial dysfunction, systemic inflammation and disruption of the immune system. The goal of this study was to characterize the PD-1/PD-L1 system, an important immune checkpoint system, on macrophages and Hofbauer cells (HBC) in the placenta of preeclamptic patients. The expression of the macrophage markers CD68 and CD163 as well as the proteins PD1 and PD-L1 in the placenta of preeclamptic patients was examined by immunohistochemistry and immunofluorescence in comparison to the placenta of healthy pregnancies. The numbers of CD68-positive and CD163-positive macrophages were significantly downregulated in the decidua (p = 0.021 and p = 0.043) and in the chorionic villi (p < 0.001 and p < 0.001) of preeclamptic patients. The majority of macrophages in the decidua and the chorionic villi were identified to be CD163-positive, indicating a predominantly M2-polarisation. The expression of PD1 on maternal macrophages of the decidua (p < 0.001) and on Hofbauer cells (p < 0.001) was shown to be significantly lower in preeclampsia. Looking at the protein PD-L1 the expression was proven to be downregulated on maternal macrophages in the decidua of preeclamptic patients (p = 0.043). This difference was only caused by a downregulation of PD-L1 expression in male offspring (p = 0.004) while there was no difference in female offspring (p = 0.841). The variation of the immune checkpoint molecules PD1 and PD-L1 in preeclampsia might play an important role in the development of inflammation seen in preeclamptic patients. It might thereby be an important target in the therapy of preeclampsia., Competing Interests: Declaration of Competing Interest N.D. reports funding from MSD, Novartis, Pfizer, Roche, AstraZeneca, TEVA, Mentor, and MCI Healthcare. C.D. is funded by Roche, AstraZeneca, TEVA, Mentor, and MCI Healthcare. All other authors declare no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The relationship between fertility and maternal body weight is shaped like an inverted "U," meaning that fertility is negatively affected in overweight or underweight women. Timely and appropriate maternal-fetal interaction is a crucial part of successful pregnancy. However, it is not clear how body weight affects maternal-fetal interaction. Placental villi are the bridge for maternal-fetal interaction. Therefore, we collected villi from pregnant women with different body mass indexes (BMI), who voluntarily underwent induced abortion, to construct a molecular network via RNA-seq. Surprisingly, based on global and significant gene network analysis, we found that dysregulation of inflammatory reaction, cell adhesion, and immune response were the most significantly enriched pathways. We also conducted dynamic gene expression analysis with BMI as a variable, and identified several distinct clusters. Among them, cluster 9 showed an inverted "U" shape and genes in it were mainly enriched in chemical synaptic transmission and cell-cell adhesion via plasma-membrane adhesion molecules. Additionally, genes in the "U" shaped cluster (cluster 5) were enriched in regulation of adaptive immune response based on somatic recombination of immune receptors built from immunoglobulin superfamily domains and negative regulation of immune response. We thus conclude that maternal body weight can affect maternal-fetal interaction through alterations or aberrant activation of inflammatory reaction and immune response. Regulating inflammatory reaction may be a potential therapeutic strategy to improve fertility of overweight and underweight people., (© 2022. The Author(s), under exclusive licence to Society for Reproductive Investigation.)

Placenta accreta is an abnormality of the placenta caused by the chorionic villi invading the muscular layer, which can cause serious bleeding, infection, shock, bladder invasion, uterine perforation, and even death. However, the etiology of placental accreta is not entirely clear. In the present study, high-throughput sequencing results showed that FYN is highly expressed in the placental accreta position in the placenta accreta group and is a key regulator of cell invasion and migration. Therefore, we aimed to evaluate the role and potential molecular mechanism of FYN in placenta accreta. The results showed that FYN was highly expressed in the placenta tissues of the placenta accreta group. Furthermore, the levels of phosphorylated STAT3, p38, and JNK in the placenta accreta group were remarkably increased compared with those in the control group. In addition, FYN knockdown considerably decreased the migration and invasion rates of trophoblast cells (HTR8/SVneo) and inhibited the levels of phosphorylated STAT3, p38, and JNK. After subsequently blocking these signaling pathways, the invasion and migration abilities of HTR8/SVneo cells were substantially decreased. In conclusion, FYN may promote excessive trophocyte cell invasion by activating STAT3, p38, and JNK pathways and can be a new target for placenta accreta prevention and treatment., Competing Interests: Conflicts of interest The authors declare no conflict of interest., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

Objective: Explored the expression of miR-29a in puerpera with RSA and its influencing mechanism. Method: 52 patients with RSA group were divided into 30 cases representing ≤3 abortions and 22 cases with ≥4 abortions,thirty healthy women who had induced abortion during the same period as the control group. The differences in the expression levels of miR-29a, FKBP52 mRNA, VEGF, MVD, and HIF-lα were compared between the groups by conducting a correlation analysis. Results: The expression levels of miR-29a, VEGF, MVD, and HIF-lα in the chorionic villus were significantly higher among patients in the group with ≥4 abortions than in those in the group with ≤3 abortions ( P < 0.05), and the expression levels of FKBP52 mRNA were lower in the former than in the latter ( P < 0.05). A Spearman correlation analysis revealed that the expression levels of miR-29a were positively correlated with the levels of VEGF, MVD, and HIF-lα ( P < 0.05) and negatively correlated with the expression level of FKBP52 mRNA ( P < 0.05). Conclusion: MiR-29a may be involved in the pathogenesis of RSA by inhibiting the protein expressions of FKBP52 and VEGF, promoting the apoptosis of trophoblasts, and impairing neovascularization, resulting in placental vascular dysplasia..

Preeclampsia is a human pregnancy-specific disease characterized by abnormal placentation that usually presents with maternal hypertension and proteinuria. The main hallmark of preeclampsia, impaired trophoblast migration, and the subsequent disruption of uterine arteries remodeling lead to several molecular alterations in the placental compartments with those occurring in the chorionic villi being of the utmost importance. Given the essential role of the endocannabinoid system during preimplantation and trophoblast migration, we have combined the histological and hyperspectral imaging analyses to shed light on the involvement of two cannabinoid receptors in the macromolecular alterations related to preeclampsia. The results obtained by immunohistochemistry showed a significant increase in the protein levels of cannabinoid receptor 1 (CB1) in the preeclamptic chorionic villi. However, no changes were reported regarding transient receptor potential vanilloid 1 (TRPV-1) levels either in the bulk placental samples or chorionic villi when comparing control and preeclamptic patients. Histological analysis and Fourier-transform infrared spectroscopy (FTIRI) showed an increase in collagen deposition together with higher levels of lipid peroxidation and phosphorylated compounds in the pathological villi. Since CB1 enhancement has been described as promoting fibrosis and oxidative stress in several tissues, we proposed that the higher receptor abundance in preeclampsia could be triggering similar molecular effects in preeclamptic term placentas.

Missed abortion (MA) is a special form of spontaneous abortion that is increasing in incidence. However, the precise molecular mechanisms underlying MA, especially regarding the decidua, are poorly understood. Herein, we identified molecular signaling pathways related to MA by comparing the decidua of women experiencing normal pregnancy and MA using a quantitative proteomics approach based on HPLC-MS/MS and iTRAQ labeling. Integrated bioinformatics analysis of villi and decidua was performed to reveal potential crosstalk signals in closely related tissues. We identified 2277 proteins with high confidence in decidua, of which 232 were differentially expressed in MA samples. Specifically, we reported that integrated quantitative proteomic and bioinformatic analysis revealed altered proteins in MA and the mechanisms underpinning MA involved numerous pathways, especially ribosome and cellular metabolism signaling. Moreover, Importin 9, Cullin 1 and COX6C are critical for MA, and their altered expression might contribute to the pathophysiology of MA. In particular, COX6C was dramatically down-regulated in both decidua and villi of MA. COX6C was also found to be highly expressed in syncytiotrophoblastic and cytotrophoblastic cells in villi and widely expressed in decidua of the control group, but dramatically decreased in the MA group. Functional analysis showed that knockdown of COX6C inhibited apoptosis process in both HTR-8 and SiHa cells, suggesting that COX6C may play protective effects in MA. Thus, this study could help to map the regulatory protein network related to MA and contribute to the pathophysiological mechanisms of MA., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Dysregulation of transcriptional programs that are tightly regulated by DNA methylation during placental and fetal development at different gestational stages, may cause recurrent miscarriage. Here, we examined genome-wide DNA methylation in chorionic villi and decidual tissues from patients suffering RM and from healthy women who had undergone artificial abortion (n = 5 each). We found that 13,426 and 5816 CpG sites were differentially methylated in chorionic villi and decidua, respectively. DNA methylation profiles of chorionic villi, but not decidua, in RM patients was clearly distinct from AA controls. Among the differentially methylated genes, the enhancer region of SPATS2L was significantly more highly methylated in RM patients (n = 19) than AA controls (n = 19; mean methylation level, 52.0%-vs.-28.9%, P < 0.001), resulting in reduced expression of SPATS2L protein in the former. Functionally, depletion of SPATS2L in extravillous trophoblast cells decreased their invasion and migration abilities. Our data indicate that particularly the chorionic villi in RM patients exhibit distinct DNA methylation profiles compared with normal pregnancies and that this changed DNA methylation status may impede the progression of embryo development via the altered expression of genes such as SPATS2L in the villi., (© 2022. The Author(s).)

Background: As a progesterone receptor antagonist, mifepristone combined with misoprostol is widely used to terminate early pregnancy in clinical practice. It has also been reported that mifepristone may cause cell death in decidual cells and result in hemorrhage of the decidua and insufficient blood supply. However, little is known about the histological effects of mifepristone on human decidua and chorion., Methods: Histological and subcellular structural changes of decidua and chorionic villi from women taking mifepristone at early pregnancy times were examined by Hematoxylin and eosin (H&E) staining and transmission Electron microscope. The expression of apoptosis-related proteins Bax/Bcl-2 was examined by immunohistochemistry., Results: After 48 h of mifepristone administration, the decidua tissue and chorionic villus structures were altered in women within 39-49 days of gestation and displayed varying degrees of degeneration and necrosis-like features. Apoptotic events were observed in the decidua and chorionic villi of early pregnancy, and mifepristone treatment significantly increases the number of apoptotic cells. The increased apoptotic events were concomitant with the increased expression of Bax and decreased expression of Bcl-2., Conclusion: This study provides evidence that mifepristone induces histological and subcellular changes in decidua and chorionic villi. Mifepristone modulates the relative ratio of Bax/Bcl-2 and the increased apoptosis contributes to the pregnancy termination at early stage of pregnancy., (© 2022. The Author(s).)

Problem: We aimed to evaluate potential biomarkers and candidate drugs for recurrent spontaneous abortion (RSA) and explore functional circular RNA pathways involved in regulating RSA., Method of Study: Expression profiles of placental villus and decidua samples derived from females with RSA and those with healthy pregnancies who underwent induced abortion were analyzed using high-throughput RNA whole transcriptome sequencing. Abnormally expressed circular RNAs in a larger cohort of samples were validated using real-time quantitative polymerase chain reaction. Drug discovery and molecular docking were performed using online databases and the Autodock tool, respectively., Results: In total, 2103 and 2160 circular RNAs were detected in three pairs of villi and three pairs of decidual tissues, respectively. A total of 22 circular RNAs, 58 miRNAs, and 393 mRNAs with significantly different expression patterns were identified. Five circular RNAs were verified, and the expression of hsa&#95;circ&#95;0088485 was significantly upregulated in the RSA group (P = .041) with a high area under the curve value (.727), sensitivity (76.5%), and specificity (64.7%). GO and KEGG enrichment analyses indicated that differentially expressed genes were associated with angiogenesis and cell adhesion. Drug discovery and molecular docking were analyzed based on 93 differentially expressed mRNAs of the ceRNA network. A total of 36 chemicals were identified as putative bioactive molecules for RSA, and one representative chemical was identified for docking with six proteins., Conclusions: These findings provide novel insights into the mechanism of regulation of RSA by circular RNA and its clinical diagnosis and treatment., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Trypanosoma cruzi crosses the placental barrier and produces the congenital transmission of Chagas disease (CD). Structural alterations of the chorionic villi by this parasite have been described in vitro, but little is known about trophoblast turnover in placentas from women with CD., Objective: To analyze the proliferation and fusion processes in placentas from women with CD., Methods: Archived human term placenta paraffin-embedded blocks were used, from women with CD (CDP), and no pathology (NP). Immunohistochemistry tests were performed for Ki67 to calculate the proliferation index (PI) of cytotrophoblast (CTB) and Syncytin-1, a fusion marker of syncytiotrophoblast (STB). Hematoxylin/Eosin stained sections were employed to analyze STB percentages, STB detachment areas and syncytial knots quantity. Non parametric Student's t-tests were performed (p < 0.05)., Results: Syncytial knots and STB detachment significantly increased in placental villi from the CDP group. STB percentage was significantly lower in the CDP group as well as the PI and Syncytin-1 expression significantly decreased in these placentas, compared with control (NP)., Conclusion: Dynamic of trophoblast turnover is altered in placentas from women with CD. These changes may lead into a gap in the placental barrier possibly allowing the parasite entry into the chorionic villi.

Objective: To investigate the expression of Talin1 in the fallopian tube and chorionic villi in patients with tubal pregnancy and its role in regulating invasion and migration of trophoblasts., Methods: Immunohistochemistry and Western blotting were used to detect the localization and expression level of Talin1 in the fallopian tube and chorionic villi in patients with tubal pregnancy and in women with normal pregnancy. In the cell experiment, HTR-8/SVneo cells was transfected with Talin1 siRNA and the changes in cell invasion and migration were assessed using scratch assay and Transwell assay. The expressions of MMP-2, MMP-9, N-cadherin and Snail in the transfected cells were detected by qRT-PCR and Western blotting., Results: Positive expression of Talin1 was detected in both normal fallopian tube tissues and tissues from women tubal pregnancy, and its expression was localized mainly in the cytoplasm of cilia cells. The expression level of Talin1 was significantly higher in both the fallopian tube and chorionic villi in women with tubal pregnancy than in normal fallopian tube and chorionic villi samples ( P < 0.01). In HTR-8/SVneo cells, transfection with Talin1 siRNA significantly inhibited cell invasion ( P < 0.01) and migration ( P < 0.05), down-regulated the expression of N-cadherin, MMP-2 and Snail ( P < 0.05), and up-regulated the expression of MMP-9 in the cells ( P < 0.05)., Conclusion: The expression of Talin1 in the fallopian tube and chorionic villi is significantly increased in women with tubal pregnancy, suggesting the association of Talin1-regulated trophoblast cell invasion with the occurrence of tubal pregnancy.

The human placenta is a rapidly developing transient organ that is key to pregnancy success. Early development of the conceptus occurs in a low oxygen environment before oxygenated maternal blood begins to flow into the placenta at ~10-12 weeks' gestation. This process is likely to substantially affect overall placental gene expression. Transcript variability underlying gene expression has yet to be profiled. In this study, accurate transcript expression profiles were identified for 84 human placental chorionic villus tissue samples collected across 6-23 weeks' gestation. Differential gene expression (DGE), differential transcript expression (DTE) and differential transcript usage (DTU) between 6-10 weeks' and 11-23 weeks' gestation groups were assessed. In total, 229 genes had significant DTE yet no significant DGE. Integration of DGE and DTE analyses found that differential expression patterns of individual transcripts were commonly masked upon aggregation to the gene-level. Of the 611 genes that exhibited DTU, 534 had no significant DGE or DTE. The four most significant DTU genes ADAM10 , VMP1 , GPR126 , and ASAH1, were associated with hypoxia-responsive pathways. Transcript usage is a likely regulatory mechanism in early placentation. Identification of functional roles will facilitate new insight in understanding the origins of pregnancy complications.

Maintenance of a healthy pregnancy is reliant on a successful balance between the fetal and maternal immune systems. Although the maternal mechanisms responsible have been well studied, those used by the fetal immune system remain poorly understood. Using suspension mass cytometry and various imaging modalities, we report a complex immune system within the mid-gestation (17-23 weeks) human placental villi (PV). Consistent with recent reports in other fetal organs, T cells with memory phenotypes, although rare in abundance, were detected within the PV tissue and vasculature. Moreover, we determined that T cells isolated from PV samples may be more proliferative after T cell receptor stimulation than adult T cells at baseline. Collectively, we identified multiple subtypes of fetal immune cells within the PV and specifically highlight the enhanced proliferative capacity of fetal PV T cells., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Preeclampsia (PE) involves inadequate placental function. This can occur due to elevated pro-inflammatory tumor necrosis factor-α (TNF-α). In other tissues, TNF-α signals via sphingosine kinase 1 (SphK1). SphK1 hinders syncytial formation. Whether this occurs downstream of TNF-α signaling is unclear. We hypothesized that placental SphK1 levels are higher in PE and elevated TNF-α decreases syncytial function, increases syncytial shedding, and increases cytokine/factor release via SphK1 activity. Term placental biopsies were analyzed for SphK1 using immunofluorescence and qRT-PCR. Term placental explants were treated after 4 days of culture, at the start of syncytial regeneration, with TNF-α and/or SphK1 inhibitors, PF-543. Syncytialization was assessed by measuring fusion and chorionic gonadotropin release. Cell death and shedding were measured by lactate dehydrogenase release and placental alkaline phosphatase-positive shed particles. Forty-two cytokines were measured using multiplex assays. Placental SphK1 was increased in PE. Increased cell death, shedding, interferon-α2, IFN-γ-induced protein 10, fibroblast growth factor 2, and platelet-derived growth factor-AA release induced by TNF-α were reversed upon SphK1 inhibition. TNF-α increased the release of 26 cytokines independently of SphK1. TNF-α decreased IL-10 release and inhibiting SphK1 reversed this effect. Inhibiting SphK1 alone decreased TNF-α release. Hence, SphK1 partially mediates the TNF-α-induced PE placental phenotype, primarily through cell damage, shedding, and specific cytokine release.

Placental function requires organized growth, transmission of nutrients, and an anti-inflammatory milieu between the maternal and fetal interface, but placental factors important for its function remain unclear. Renalase is a pro-survival, anti-inflammatory flavoprotein found to be critical in other tissues. We examined the potential role of renalase in placental development. PCR, bulk RNA sequencing, immunohistochemistry, and immunofluorescence for renalase and its binding partners, PMCA4b and PZP, were performed on human placental tissue from second-trimester and full-term placentas separated into decidua, placental villi and chorionic plates. Quantification of immunohistochemistry was used to localize renalase across time course from 17 weeks to term. Endogenous production of renalase was examined in placental tissue and organoids. Renalase and its receptor PMCA4b transcripts and proteins were present in all layers of the placenta. Estimated RNLS protein levels did not change with gestation in the decidual samples. However, placental villi contained more renalase immunoreactive cells in fetal than full-term placental samples. RNLS co-labeled with markers for Hofbauer cells and trophoblasts within the placental villi. Endogenous production of RNLS, PMCA4b, and PZP by trophoblasts was validated in placental organoids. Renalase is endogenously expressed throughout placental tissue and specifically within Hofbauer cells and trophoblasts, suggesting a potential role for renalase in placental development and function. Future studies should assess renalase's role in normal and diseased human placenta., (© 2022. The Author(s).)

Recurrent spontaneous abortion (RSA) effects both the physical and mental health of women of reproductive age. Trophoblast dysfunction may result in RSA due to shallow placental implantation. The mechanisms underlying formyl peptide receptor 2 (FPR2) on the biological functions of trophoblasts remain to be elucidated. The present study aimed to explore the potential functions of FPR2, a G protein‑coupled receptor, in placental trophoblasts. The location and expression levels of FPR2 in the villi tissue of patients with RSA were detected using immunohistochemical staining, reverse transcription‑quantitative PCR and western blotting. Following the transfection of small interfering RNA targeting FPR2 in HTR‑8/SVneo cells, a Cell Counting Kit‑8 assay was used to determine the levels of cell viability. Flow cytometry was used to examine the levels of cell apoptosis and gap closure and Transwell assays were carried out to evaluate the levels of cell migration and invasion. A tube formation assay was performed to detect the levels of capillary‑like structure formation. Western blotting was used to detect the expression levels of proteins in the associated signaling pathways. The expression of FPR2 was present in villi trophoblasts and was markedly increased in patients with RSA. The levels of trophoblast invasion, migration and tube formation were markedly increased following FPR2 knockdown, whereas the levels of apoptosis were markedly decreased. In addition, FPR2 knockdown caused an increase in the phosphorylation levels of AKT and PI3K. Thus, FPR2 may be involved in the regulation of trophoblast function via the PI3K/AKT signaling pathway. The results of the present study provided a theoretical basis for the use of FPR2 as a target for the treatment of trophoblast‑associated diseases, such as RSA.

Preeclampsia is a significant contributor for maternal or fetal morbidity and mortality, which is characterized by reduced invasion capacity of trophoblasts and is regulated by extracellular matrix (ECM). It is still under investigation whether chorionic villus-derived mesenchymal stem cells (CVMSC) could affect the functionality of trophoblasts. In this study, CVMSC-derived exosomes were isolated; their effect on trophoblasts was investigated based on the CCK8 assay, migration assay, and apoptosis detection. And the underlying mechanism of this effect was investigated using mRNA sequencing, western blot, co-immunoprecipitation, luciferase report assay, and ubiquitination assay. The results show that CVMSC-derived exosomes promote migration and proliferation of trophoblasts, and also reduce cell apoptosis. mRNA sequencing confirmed that after treatment of CVMSC-derived exosomes, Tripartite Motif Containing 72 (TRIM72) expression was upregulated and Tumor Protein P53 (P53) expression was downregulated, both significantly in trophoblasts. Subsequent study confirms that TRM72 can directly interact with P53 and promote P53 ubiquitination and proteasomal degradation, reducing apoptosis rate and elevating proliferation and migration in trophoblasts. Our study confirms that CVMSC-derived exosomes promote trophoblast migration and proliferation by upregulating TRIM72 expression, and subsequently advance P53 ubiquitination and proteasomal degradation., (© 2021 Federation of American Societies for Experimental Biology.)

We performed a comparative analysis of the expression of fucosylated glycans and morphometric characteristics of the terminal villi of the placenta, depending on the severity of preeclampsia (PE). Similar patterns of the expression of fucosylated glycans in the syncytiotrophoblast glycocalyx were revealed in the placental tissue of patients with normal pregnancy and with mild and severe PE: predominance of glycans with α1,6-fucose in the core, clustered fucose residues, and LeX glycan over α1,2-fucose-containing glycans. The expression pattern of fucosylated glycans and the composition of the endothelial glycocalyx are normally close to the expression pattern and composition of the syncytiotrophoblast glycocalyx; in case of mild and severe PE, the expression pattern of fucosylated glycans was changed uniformly, and α1,2-fucose-containing glycans significantly prevailed in the endothelial glycocalyx. According to the results of Fisher's LSD test, in patients with severe PE, the total vascular area in the villus prevailed over the indices established during physiological course of pregnancy (p=0.04) and mild PE (p=0.04). Correlation analysis revealed direct and reciprocal relationships between the morphometric characteristics of the terminal villi of the placenta and the expression of fucosylated glycans in the syncytiotrophoblast and endothelium in PE. Our results indicate a changed expression of fucosylated glycans in the glycocalyx of placental barrier structures and the morphometric parameters of villi in PE of different severity, which can affect the function of the placental barrier., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

The placenta is a fetal-derived organ whose function is crucial for both maternal and fetal health. The human placenta contains a population of fetal macrophages termed Hofbauer cells. These macrophages play diverse roles, aiding in placental development, function and defence. The outer layer of the human placenta is formed by syncytiotrophoblast cells, that fuse to form the syncytium. Adhered to the syncytium at sites of damage, on the maternal side of the placenta, is a population of macrophages termed placenta associated maternal macrophages (PAMM1a). Here we discuss recent developments that have led to renewed insight into our understanding of the ontogeny, phenotype and function of placental macrophages. Finally, we discuss how the application of new technologies within placental research are helping us to further understand these cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Thomas, Naidu, Appios and McGovern.)

Recurrent spontaneous abortion (RSA) affects 5% of childbearing-age women worldwide. Inadequate trophoblast invasion is one of the main reasons for the development of RSA; however, the underlying molecular mechanisms for RSA have not been fully understood, and further explanation is urgently needed. C-X-C motif chemokine ligand 5 (CXCL5) is reported to contribute to the invasion of cancer cells, and its aberrant expression is associated with the cellular process of tumor pathology. Considering the high behavioral similarity between trophoblast cells and cancer cells, we hypothesized that CXCL5 may influence trophoblast invasion, and its expression levels in villous tissue may be correlated with RSA. In this study, we firstly investigated the CXCL5 expression in placental villous tissues of 15 RSA patients and 13 control patients, and the results showed that CXCL5 levels were significantly lower in villous tissue from RSA patients than those of the controls. Further in vitro experiments presented that recombinant human CXCL5 can enhance trophoblast migration, invasion, and epithelial-to-mesenchymal transition (EMT) process. We also demonstrated that CXCL5 exerted these effects on trophoblast cells through PI3K/AKT/ERK1/2 signaling pathway. In conclusion, these data indicate that CXCL5 downregulation in human villous tissue is correlated with RSA. Additionally, we found that estrogen, progesterone, human chorionic gonadotropin, and decidual stromal cells can regulate CXCL5 and chemokine receptor 2 (CXCR2) expression of trophoblast in a cell manner., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zhang, Ding, Wang, Yin, Zhang and Yang.)

Purpose: In this study, we evaluated the feasibility of the combining CNV-seq and quantitative fluorescence polymerase chain reaction (QF-PCR) for miscarriage analysis in clinical practice., Methods: Over a 35-month period, a total of 389 fetal specimens including 356 chorionic villi and 33 fetal muscle tissues were analyzed by CNV-seq and QF-PCR. Relationships between the risk factors (e.g., advanced maternal age, abnormal pregnancy history, and gestational age) and incidence of these chromosomal abnormalities were further analyzed by subgroup., Results: Clinically significant chromosomal abnormalities were identified in 58.95% cases. Aneuploidy was the most common abnormality (46.84%), followed by polyploidy (8.16%) and structural chromosome anomalies (3.95%). In sub-group analysis, significant differences were found in the total frequency of chromosomal abnormalities between the early abortion and the late abortion group, as well as in the distribution of chromosomal abnormalities between the advanced and the younger maternal age group. Meanwhile, the results of the logistic regression analysis identified a trend suggesting that the percentage of fetal chromosomal abnormalities is significantly higher in advanced maternal age, lesser gestational age, and lesser number of prior miscarriages., Conclusion: Our study suggests that CNV-seq and QF-PCR are efficient and reliable technologies in the fetal chromosome analysis of miscarriages and could be used as a routine selection method for the genetic analysis of spontaneous abortion., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: B7-H4, a checkpoint molecule of the B7 family, regulates a broad spectrum such as T-cell activation, cytokine secretion, tumour progression, and invasion capacities. Our previous data revealed that soluble B7-H4 (sB7-H4) blood serum levels are elevated in women at high risk for the hypertensive pregnancy disorder preeclampsia (PE) in the first trimester, as well as in patients with confirmed early/late-onset PE., Aim: We here aim to investigate the expression pattern of B7-H4 in placental tissues of PE and HELLP Syndrome versus control group., Methods: B7-H4 protein expression and localization were investigated by immunoblotting and co-immunohistochemistry in placental chorionic villous and decidual basalis tissues., Results: B7-H4 protein was prominently expressed at the cell membrane, in the cytoplasm of the syncytiotrophoblast (STB) and interstitial extravillous trophoblast (EVT). B7-H4 protein levels in placental chorionic villous tissue were significantly higher in women with early-onset/late-onset PE and HELLP, while it was decreased in decidual basalis tissues of early-onset PE and HELLP compared with controls., Conclusion: B7-H4 was inversely expressed in placental chorionic villous and decidual basalis tissues of PE and HELLP patients. The increase in B7-H4 in the STB in PE and HELLP may lead to excessive apical expression and release of soluble B7-H4 in the maternal circulation. In contrast, the decrease in B7-H4 in decidual basalis tissues could be related to the decrease in invasion ability of the EVT in PE. Thus, the current results strongly suggest that B7-H4 is involved in the pathogenesis of PE and HELLP., (© 2021 The Authors. American Journal of Reproductive Immunology published by John Wiley & Sons Ltd.)

Long noncoding RNAs (lncRNAs) have been reported to be involved in various cellular processes and to participate in a variety of human diseases. Recently, increasing studies have reported that lncRNAs are related to many reproductive diseases, such as pathogenesis of recurrent pregnancy loss (RPL), preeclampsia (PE) and gestational diabetes mellitus (GDM). In this study, we aimed to investigate the effect of LINC01088 in trophoblast cells and its potential role in pathogenesis of RPL. LINC01088 was found to be upregulated in first-trimester chorionic villi tissues from RPL patients. Increased LINC01088 repressed proliferation, migration and invasion of trophoblast cells, and promoted apoptosis of trophoblast cells. Further exploration indicated that LINC01088 decreased the production of nitric oxide (NO) by binding and increasing Arginase-1 and decreasing eNOS protein levels. Importantly, JNK and p38 MAPK-signaling pathways were active after overexpression of LINC01088. In conclusion, our studies demonstrated that LINC01088 plays an important role in the pathogenesis of RPL, and is a potential therapeutic target for the treatment of RPL., (© The Author(s) 2021. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Chronic venous disease (CVD) is the response to a series of hemodynamic changes in the venous system and the onset of this disease is often triggered by pregnancy. Placental tissue is particularly sensitive to the characteristic changes which occurs in venous hypertension. In this regard, changes in the extracellular matrix (ECM), that occur to adapt to this situation, are fundamental to controlling elastogenesis. Therefore, the aim of the present study was to analyze the changes that occur in the mRNA and protein expression level of proteins related to elastogenesis in the placental villi of women diagnosed with CVD, in the third trimester of pregnancy. An observational, analytical and prospective cohort study was conducted, in which the placenta from 62 women with CVD were compared with that in placenta from 52 women without a diagnosis of CVD. Gene and protein expression levels were analyzed using reverse transcription‑quantitative PCR and immunohistochemistry, respectively. The results showed a significant decrease in the gene and protein expression level of EGFL7 in the placental villi of women with CVD. By contrast, significant increases in the gene and protein expression level of ECM‑related proteins, such as tropoelastin, fibulin 4, fibrillin 1 and members of the lysyl oxidase family (LOX and LOXL‑1) were also found in the placental villi of women with CVD. To the best of our knowledge, the results from the present study showed for the first time that CVD during pregnancy was associated with changes in the mRNA and protein expression level in essential components of the EGFL7‑modulated elastogenesis process in placental villi.

During pregnancy, freely floating placental villi are adapted to fluid shear stress due to placental perfusion with maternal plasma and blood. In vitro culture of placental villous explants is widely performed under static conditions, hoping the conditions may represent the in utero environment. However, static placental villous explant culture dramatically differs from the in vivo situation. Thus, we established a flow culture system for placental villous explants and compared commonly used static cultured tissue to flow cultured tissue using transmission and scanning electron microscopy, immunohistochemistry, and lactate dehydrogenase (LDH) and human chorionic gonadotropin (hCG) measurements. The data revealed a better structural and biochemical integrity of flow cultured tissue compared to static cultured tissue. Thus, this new flow system can be used to simulate the blood flow from the mother to the placenta and back in the most native-like in vitro system so far and thus can enable novel study designs.

A comparative morphological study was carried out to analyze the number of syncytial knots and VEGF expression in placental villi in parturient women with COVID-19 categorized by the disease severity. The number of syncytial knots was assessed on specimens stained with hematoxylin and eosin. VEGF expression was determined by immunohistochemical analysis in syncytiotrophoblast and villous endothelial cells. Morphological study of the placenta tissue of parturient women with COVID-19 showed increased numbers of syncytial knots in the villi, indicating the development of preplacental hypoxia. High VEGF expression in syncytiotrophoblast and vascular endotheliocytes reflects a stereotyped response to hypoxia and can underlie the development of a preeclampsia-like syndrome. The number of syncytial knots and VEGF expression in placental villi in parturient women with COVID-19 depended on the disease severity., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Epitope H contains an O-linked N-acetylglucosamine (O-GlcNAcH) residue in a specific conformation and/or environment recognized by the mouse monoclonal antibody H. O-GlcNAcH is present in several types of cells and in several polypeptides, including cytokeratin 8 and vimentin, on the latter in cells under stress. In the present work, we examined the expression of the O-GlcNAcH in 60 cases of endometrial curettings from missed miscarriage cases containing normal and simple hydropic degenerated chorionic villi in each case, using monoclonal antibody H and indirect immunoperoxidase and Western blot immunoblot. In all cases examined the expression of the O-GlcNAcH was cytoplasmic as follows: (1) syncytiotrophoblastic cells showed very low expression in chorionic villi (CV) with nonhydropic degeneration (NHD) and high expression in hydropic degenerated (HD) CV; (2) cytotrophoblastic cells showed low expression in CV with NHD and high expression in HD CV; (3) fibroblastic cells showed high expression in CV with NHD and very low expression in HD CV; (4) histiocytes showed very low expression in both types of CV; (5) endothelial cells showed high expression in both types of CV. An immunoblot of CV from one case of a legal abortion from a normal first-trimester pregnancy showed 5 polypeptides with 118.5, 106.3, 85, 53, and 36.7 kD bearing the epitope H and the 53 kD corresponded to cytokeratin 8. The expression of the O-GlcNAcH is upregulated in the trophoblastic cells and downregulated in the fibroblastic cells in the HD CV in comparison to the NHD CV., Competing Interests: The authors declare no conflict of interest., (Copyright © 2020 by the International Society of Gynecological Pathologists.)

Introduction: Placental oxidative stress features in pregnancy pathologies but in clinical trials antioxidant supplementation has not improved outcomes. N-acetylcysteine (NAC) stimulates glutathione production and is proposed as a therapeutic agent in pregnancy. However, key elements of N-acetylcysteine biology, including its cellular uptake mechanism, remains unclear. This study explores how the cystine/glutamate transporter xCT may mediate N-acetylcysteine uptake and how N-acetylcysteine alters placental redox status., Methods: The involvement of xCT in NAC uptake by the human placenta was studied in perfused placenta and Xenopus oocytes. The effect of short-term N-acetylcysteine exposure on the placental villous proteome was determined using LC-MS. The effect of N-acetylcysteine on Maxi-chloride channel activity was investigated in perfused placenta, villous fragments and cell culture., Results: Maternoplacental N-acetylcysteine administration stimulated intracellular glutamate efflux suggesting a role of the exchange transporter xCT, which was localised to the microvillous membrane of the placental syncytiotrophoblast. Placental exposure to a bolus of N-acetylcysteine inhibited subsequent activation of the redox sensitive Maxi-chloride channel independently of glutathione synthesis. Stable isotope quantitative proteomics of placental villi treated with N-acetylcysteine demonstrated changes in pathways associated with oxidative stress, apoptosis and the acute phase response., Discussion: This study suggests that xCT mediates N-acetylcysteine uptake into the placenta and that N-acetylcysteine treatment of placental tissue alters the placental proteome while regulating the redox sensitive Maxi-chloride channel. Interestingly N-acetylcysteine had antioxidant effects independent of the glutathione pathway. Effective placental antioxidant therapy in pregnancy may require maintaining the balance between normalising redox status without inhibiting physiological redox signalling., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Congenital toxoplasmosis is represented by the transplacental passage of Toxoplasma gondii from the mother to the fetus. Our studies demonstrated that T. gondii developed mechanisms to evade of the host immune response, such as cyclooxygenase (COX)-2 and prostaglandin E 2 (PGE 2 ) induction, and these mediators can be produced/stored in lipid droplets (LDs). The aim of this study was to evaluate the role of COX-2 and LDs during T. gondii infection in human trophoblast cells and villous explants. Our data demonstrated that COX-2 inhibitors decreased T. gondii replication in trophoblast cells and villous. In BeWo cells, the COX-2 inhibitors induced an increase of pro-inflammatory cytokines (IL-6 and MIF), and a decrease in anti-inflammatory cytokines (IL-4 and IL-10). In HTR-8/SVneo cells, the COX-2 inhibitors induced an increase of IL-6 and nitrite and decreased IL-4 and TGF-β1. In villous explants, the COX-2 inhibitors increased MIF and decreased TNF-α and IL-10. Furthermore, T. gondii induced an increase in LDs in BeWo and HTR-8/SVneo, but COX-2 inhibitors reduced LDs in both cells type. We highlighted that COX-2 is a key factor to T. gondii proliferation in human trophoblast cells, since its inhibition induced a pro-inflammatory response capable of controlling parasitism and leading to a decrease in the availability of LDs, which are essentials for parasite growth.

Dysregulated extravillous trophoblast invasion and proliferation are known to increase the risk of recurrent spontaneous abortion (RSA); however, the underlying mechanism remains unclear. Herein, in our retrospective observational case-control study we show that villous samples from RSA patients, compared to healthy controls, display reduced succinate dehydrogenase complex iron sulfur subunit (SDHB) DNA methylation, elevated SDHB expression, and reduced succinate levels, indicating that low succinate levels correlate with RSA. Moreover, we find high succinate levels in early pregnant women are correlated with successful embryo implantation. SDHB promoter methylation recruited MBD1 and excluded c-Fos, inactivating SDHB expression and causing intracellular succinate accumulation which mimicked hypoxia in extravillous trophoblasts cell lines JEG3 and HTR8 via the PHD2-VHL-HIF-1α pathway; however, low succinate levels reversed this effect and increased the risk of abortion in mouse model. This study reveals that abnormal metabolite levels inhibit extravillous trophoblast function and highlights an approach for RSA intervention.

Inadequate trophoblast proliferation, shallow invasion and exaggerated rate of trophoblast apoptosis are implicated in early recurrent miscarriage (ERM). However, the mechanistic bases of this association have not been fully established. We aimed at investigating the involvement of fascin, an actin-bundling protein, in trophoblast activities and ERM. We found that fascin was downregulated in the cytotrophoblasts (CTBs) and distal cytotrophoblasts (DCTs) of ERM placentae. Knockdown of fascin altered cellular and nucleolar morphology, and inhibited the proliferation but increased apoptosis of trophoblastic HTR8/SVneo cells. Furthermore, fascin knockdown decreased the expression of transcription factors such as Snail1/2, Twist and Zeb1/2, mesenchymal molecules such as Vimentin and N-cadherin, and the protein expression of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphorylates signal transducer and activator of transcript 3 (STAT3). Exposure of HTR-8/SVneo cells to hypoxia reoxygenation (H/R) decreased fascin expression to affect the cells' invasion. Our results indicate for the first time that the downregulation of fascin is involved in the pathogenesis of early recurrent miscarriage; and hence a potential therapeutic target against the disease., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Transmembrane integrin receptors mediate cell-extracellular matrix as well as cell-cell adhesion. As placental trophoblast cells undergo differentiation they display changes in integrin expression or switching, but the mechanism(s) of integrin activation that supports this differentiation is still unknown. The Fermitin family of adapter proteins (FERMT 1-3) are integrin activators that mediate integrin-mediated signaling. In this study, we examined the spatiotemporal pattern of expression of FERMT1 in human chorionic villi throughout gestation and its role in HTR8-SVneo substrate adhesion and invasion. Placental villous tissue was obtained from patients undergoing elective terminations at weeks 8-14, as well as from term deliveries at weeks 37-40 and analyzed by immunofluorescence. Additionally, HTR8-SVneo trophoblast cells were transfected with FERMT1-specific siRNA or non-targeting siRNA (control) and used in cell-substrate adhesion as well as invasion assays. FERMT1 was primarily localized to membrane-associated regions at the base or around the periphery of the villous cytotrophoblast and proximal as well as distal cell column trophoblast. FERMT1 was also localized to endothelial cells of blood vessels in chorionic villi. siRNA-mediated depletion of FERMT1 in HTR8-SVneo cells did not markedly alter HTR8-SVneo cell-substrate adhesion but did significantly decrease invasion (P < 0.05) compared to control cells. These novel findings identify the presence of the integrin activator FERMT1 in trophoblast cells and that FERMT1 can regulate HTR8-SVneo cell invasion. FERMT1 may directly influence integrin activation and the subsequent integrin-mediated signaling and differentiation that underlies the acquisition of the invasive trophoblast phenotype in vivo.