Characterization of the Primary Human Trophoblast Cell Secretome Using Stable Isotope Labeling With Amino Acids in Cell Culture

The placental villus syncytiotrophoblast, the nutrient-transporting and hormone-producing epithelium of the human placenta, is a critical regulator of fetal development and maternal physiology. However, the identities of the proteins synthesized and secreted by primary human trophoblast (PHT) cells remain unknown. Stable Isotope Labeling with Amino Acids in Cell Culture followed by mass spectrometry analysis of the conditioned media was used to identify secreted proteins and obtain information about their relative rates of synthesis in syncytialized multinucleated PHT cells isolated from normal term placental villus tissue (n = 4/independent placenta). A total of 1,344 proteins were identified, most of which have not previously been reported to be secreted by the human placenta or trophoblast. The majority of secreted proteins are involved in energy and carbon metabolism, glycolysis, biosynthesis of amino acids, purine metabolism, and fatty acid degradation. Histone family proteins and mitochondrial proteins were among proteins with the slowest synthesis rate whereas proteins associated with signaling and the plasma membrane were synthesized rapidly. There was a significant overlap between the PHT secretome and proteins known be secreted to the fetal circulation by the human placenta in vivo. The generated data will guide future experiments to determine the function of individual secreted proteins and will help us better understand how the placenta controls maternal and fetal physiology.

Down‐regulation of the Sp1 transcription factor by an increase of microRNA-4497 in human placenta is associated with early recurrent miscarriage

Background The pathophysiological mechanism of recurrent miscarriage (RM) is unclear. The goals of this study were to determine the role of microRNA-4497 overexpression in placental villus tissues in early RM; To identify the potential target mRNAs of miRNA-4497; And to investigate the microRNA-4497-mediated regulatory mechanisms in placental trophoblasts. Methods Bioinformatics analysis was performed to identify the candidate target genes of miRNA-4497. The protein expression of Sp1 transcription factor (SP1), chemokine (C-X-C motif) receptor 5 (CXCR5) and bone morphogenetic protein 8a (BMP8A) were determined in the villus tissues of the RM and normal groups by Western blotting and immunohistochemistry. Cultured 293T cells were co-transfected with the miRNA-4497 agomir or luciferase reporter vectors containing the wild-type or mutant 3’-UTRs of the target mRNAs to verify the regulatory role of miRNA-4497. Results Bioinformatics analysis suggested that SP1, CXCR5 and BMP8A mRNAs are potential targets of miRNA-4497. The expression of SP1, CXCR5 and BMP8A proteins in the chorionic villus tissues of RM placentas were significantly decreased compared to those in the normal controls. Moreover, SP1 protein levels were inversely correlated with the levels of miRNA-4497 in the placentas of RM patients and normal controls. The expression of SP1 mRNA and protein were down-regulated in HTR-8/SVneo cells after forced overexpression of the miRNA-4497 agomir. The results of the co-transfection assay showed that mutation of the miRNA-4497-binding sites in the 3’-untranslated region (3’-UTR) of SP1 led to a recovery of luciferase activity upon overexpression of miRNA-4497, suggesting that SP1 could be a direct target of miRNA-4497. Conclusions An increased miRNA-4497 level in the placental villus tissues associated with recurrent miscarriage may down-regulate SP1 expression. The negative regulation of SP1 by miRNA-4497 may potentially contribute to the pathogenesis of recurrent miscarriage through promotion of trophoblast apoptosis. These findings provide novel information on the regulation of placental trophoblast apoptosis, and could be useful for the development of new therapeutic strategies for better management of recurrent miscarriage.

Perinatal outcome in abnormal placental villus proliferation and mesenchymal dysplasia

Placental mesenchymal dysplasia (PMD) is a rare vascular anomaly which is characterized by mesenchymal stem villous hyperplasia and placentomegaly. Since the modality of treatment changes it is necessary to distinguish PMD from molar pregnancy, placenta mosaicism, chorioangioma, twin pregnancy with co-existent molar pregnancy. On reviewing cases of abnormal placental villus proliferation having features of placental mesenchymal hyperplasia placentomegaly and cystic appearance of placenta in database of our hospital from 2015-2019, we reported 4 cases of abnormal placental villous proliferation. And performed systematic review of existing literature. Provisional diagnosis of PMD was made as USG and placental morphology showed 30-60% of the placenta with cystic vesicles, placentomegaly with a normal growing fetus. PMD an uncommon vascular anomaly which resembles molar pregnancy but prognosis is different. The fetus was normal in majority of the cases. This clinical entity should be kept in mind to avoid unnecessary termination of pregnancy.

MNSFβ Promotes the Proliferation and Migration of Human Extravillous Trophoblast Cells and the Villus Expression Level of MNSFβ Is Decreased in Recurrent Miscarriage Patients

<b><i>Aims:</i></b> The invasion of extravillous trophoblast (EVT) cells into maternal decidua is essential for the establishment and maintenance of pregnancy. Derangement of EVT cell invasion might cause pregnancy complications including recurrent miscarriage (RM). We previously reported that deficiency of monoclonal nonspecific suppressor factor beta (MNSFβ) led to the early pregnancy failure in mice and the decidual MNSFβ expression level in RM patients was significantly decreased, but the underlying molecular mechanism of the role that MNSFβ played at the maternal-fetal interface remains unclear. Thus, in the present study, we determined effects of downregulated MNSFβ expression on human EVT cell activities. <b><i>Methods:</i></b> The MNSFβ expression in first-trimester human decidual and placental villus tissues was detected, respectively, by immunofluorescence or immunohistochemical analyses. The MNSFβ expression level in the immortalized first-trimester human EVT cell line HTR8/SVneo was downregulated by transfecting the small interfering RNA against MNSFβ and upregulated by transfecting the recombinant pDsRed-MNSFβ plasmids. The proliferation, migration, invasion, and apoptosis activities of HTR8/SVneo cells were, respectively, determined by cytometry assay, scratch test, transwell assay, and FITC/PI staining. The expression levels of P53, RhoA, Bcl-2, Bax, and MMP-9 in HTR8/SVneo cells, as well as the expression levels of MNSFβ and RhoA in placental villi of RM patients and physically normal pregnant women (NP), were examined by Western blot analysis. <b><i>Results:</i></b> MNSFβ protein signals were observed in first-trimester human villus and extravillous trophoblast cells. The downregulated MNSFβ expression significantly attenuated the proliferation, migration, and invasion abilities of HTR8/SVneo cells, accompanied with the obviously decreased expression levels of P53, RhoA, Bcl-2, Bax, and MMP-9, whereas the upregulated MNSFβ expression in HTR8/SVneo cells represented the inverse effects. Furthermore, expression levels of MNSFβ and RhoA in first-trimester human placental villus tissues of RM patients were significantly decreased compared to that of NP women. <b><i>Conclusion:</i></b> These data suggested that MNSFβ promotes proliferation and migration of human EVT cells, probably via the P53 signaling pathway, and the deficiency of MNSFβ in placental villi might lead to early pregnancy loss by reducing proliferation and invasion activities of EVTs.