The villous trophoblast cells are of fundamental importance because they fulfill a variety of functions that are vital for the growth of the fetus and the maintenance of pregnancy. A simple in vitro villous trophoblast cell model that grows on standard tissue culture plates has been utilized for various functional studies on villous trophoblast cells. Despite the potential value of incorporating electron microscopy analysis in reports on functional analysis of primary human trophoblast cells, electron microscopy analysis is exclusively ancillary to functional analysis in previous publications. In the context of autophagy research of villous trophoblast cells using primary trophoblast cells, a detailed ultrastructural analysis of autophagy flux using electron microscopy is imperative; however, it has not been conducted to date. In this study, we isolated term villous trophoblast cells (i.e., cytotrophoblast cells, CTB cells) using the most up-to-date isolation method for isolating pure CTB cells from human term placenta and investigated the ultrastructural dynamic process of autophagy of cultured CTB cells by means of transmission electron microscopy. The initial 6 h culture resulted in CTB cell aggregation; however, the majority of CTB cells did not differentiate into syncytial cells. In contrast, after 72 h, CTB cells exhibited a promotion of differentiation into syncytial cells. The electron microscopy analysis revealed the upregulation of autophagy and visualized unique autophagic profiles during differentiation into syncytial cells, which exhibited perinuclear accumulation of extremely large autophagosomes/autolysosomes. This study provides novel insights into the reproductive biology of primary trophoblast cells, thereby demonstrating the substantial value of primary trophoblast cells as research resources.

A 10-year-old female Papillon dog that had previously developed a mammary tumor was admitted for treatment of a hypoglycemic attack. Blood examination showed severe hypoglycemia and decreased blood insulin concentration. Computed tomography indicated multiple tumors in the cranial and caudal lobes of the right lung. These tumors were resected surgically and diagnosed as pulmonary adenocarcinomas by histopathologic examination. Hypoglycemia was temporarily improved after the resection, but a hypoglycemic event occurred 2 months after the surgery. Immunohistochemistry of the tumor demonstrated the expression of insulin-like growth factor 2 in tumor cells. Western blot analysis revealed the expression of high-molecular-weight (big)–insulin-like growth factor 2 in the tumor region. Insulin-like growth factor 2 mRNA expression was also confirmed in the tumor using reverse transcription–polymerase chain reaction. These findings indicate the diagnosis of non–islet cell tumor-induced hypoglycemia caused by big-insulin-like growth factor 2 produced by the tumor in the dog. This report provides information on differentiating tumors that cause paraneoplastic hypoglycemia.

In patients with high-risk prostate cancer (HRPC) after radical prostatectomy (RP), biochemical recurrence (BCR) increases the risk of distant metastasis. Accordingly, additional prognostic biomarkers are required to identify the subpopulation of patients with HRPC who develop clinical recurrence (CR) after BCR. The objective of this study was to identify biomarkers in formalin-fixed paraffin-embedded (FFPE) RP samples that are prognostic for CR in patients with HRPC who experience BCR after RP (post-RP BCR). First, we performed a preliminary RNA sequencing analysis to comprehensively profile RNA expression in FFPE RP samples obtained from patients with HRPC who developed CR after post-RP BCR and found that many snRNAs were very abundant in preserved FFPE samples. Subsequently, we used quantitative polymerase chain reaction (qPCR) to compare the expression levels of highly abundant snRNAs in FFPE RP samples from patients with HRPC with and without CR after post-RP BCR (21 CR patients and 46 non-CR patients who had more than 5 years of follow-up after BCR). The qPCR analysis revealed that the expression levels of snRNA RNU1-1/1-2 and RNU4-1 were significantly higher in patients with CR than in patients without CR. These snRNAs were significantly correlated with clinical recurrence-free survival (RFS) in patients with HRPC who experienced post-RP BCR. Furthermore, snRNA RNU1-1/1-2 could serve as an independent prognostic factor for clinical RFS in post-RP BCR of HRPC cases where known prognostic factors (e.g., Gleason score) cannot distinguish between CR and non-CR patients. Our findings provide new insights into the involvement of snRNAs in prostate cancer progression.

Small extracellular vesicles (small EVs) play pivotal roles in intercellular communication and pregnancy maintenance, but their clinical significance in preeclampsia (PE) remains unclear. We obtained plasma samples from non-pregnant women, healthy pregnant women, and patients with early-onset (EoPE) and late-onset PE (LoPE). Small EVs were isolated using ultracentrifugation and validated using transmission electron microscopy and nanoparticle tracking analysis; in addition, Western blotting was performed to identify suitable surface markers for plasma-derived small EVs. In our analysis, we consistently detected cluster of differentiation 9 (CD9), whereas classical markers such as cluster of differentiation 63 (CD63) and tumor susceptibility gene 101 (TSG101) were absent. In a prospective, nested case–control study, we analyzed first-trimester samples by using a CD9-based ELISA for small-EV quantification. The number of small EVs did not significantly differ between non-pregnant and healthy pregnant women regardless of the gestational age. However, EVs were significantly elevated in both EoPE (3.5-fold) and LoPE (1.5-fold) compared with matched controls. First-trimester EV levels did not show differences between women who later developed PE and normal controls. These findings indicate that CD9 is a promising marker for plasma-derived small EVs and that an elevated number of small EVs is associated with established PE but has limited predictive value in early pregnancy. Further studies are required to elucidate the cellular origin and clinical implications of small EVs in PE.

Background: Small extracellular vesicles (small EVs) play pivotal roles in intercellular communication and pregnancy maintenance. Their clinical significance in preeclampsia (PE) remains unclear. Methods: Plasma samples were obtained from non-pregnant women, healthy pregnant women, and patients with early-onset (EoPE) and late-onset PE (LoPE). Small EVs were isolated by ultracentrifugation and validated using transmission electron microscopy and nanoparticle tracking analysis. Western blotting was performed to identify suitable surface markers for plasma-derived small EVs. The quantification was conducted using a cluster of differentiation 9 (CD9)-based ELISA. A prospective nested case-control study analyzed >1,400 first-trimester samples. Results: CD9 was consistently detected in plasma-derived small EVs, whereas classical markers such as cluster of differentiation 63 (CD63) and tumor susceptibility gene 101 (TSG101) were absent. the number of small EVs did not significantly differ between non-pregnant and healthy pregnant women regardless of the gestational age. However, EVs were significantly elevated in both EoPE (3.5-fold) and LoPE (1.5-fold) compared with matched controls. First-trimester EV levels did not show the difference between women who later developed PE and normal controls. Conclusions: CD9 is a promising marker for plasma-derived small EVs. Elevated numbers of small EVs are associated with established PE but show limited predictive value in early pregnancy. Further studies are required to elucidate the cellular origin and clinical implications of small EVs in PE.