Exporting Proteins Associated with Senescence Repair via Extracellular Vesicles May Be Associated with Early Pregnancy Loss.

INTRODUCTION: Dysfunction of placental development is involved in early pregnancy loss. Senescent changes have been seen in missed miscarriage, one type of pregnancy loss. Extracellular vesicles (EVs) have been widely implicated in the pathogenesis of diseases. In this study, we investigated the protein profiles in placental EVs derived from missed miscarriage in comparison with healthy pregnancy. We also investigated whether cargos packed into EVs are involved in the dysfunctional development of the placenta seen in missed miscarriage. METHODS: Proteomic analysis of placental EVs derived from healthy and missed-miscarriage placentae was performed. Three senescence-repair-associated proteins, replication protein A-70 (RPA-70), proteasome activator subunit-4 (PMSE-4), and protein activated kinase-2, (PAK-2) were examined in placental EVs and placentae, and in placental explants that had been treated with or without GW4869, by western blotting and immunohistochemistry. RESULTS: The total number of proteins associated with placental EVs was not different between the two groups. However, there were 106 and 151 abundantly expressed proteins associated with placental micro- or nano-EVs from missed miscarriage in comparison with EVs from controls. Of these abundant proteins, 59 and 81 proteins in placental micro- or nano-EVs, respectively, are associated with DNA damage/repair and cell death/survival. We further found higher levels of three senescence-repair-associated proteins (RPA-70, PMSE-4, and PAK-2) associated with placental EVs, but lower levels of these proteins in missed-miscarriage placentae. Regarding inhibition of EV formation or release by GW4869, we found that the expression of these three proteins was higher in GW4869-treated placental explants from missed miscarriage. DISCUSSION: Our data may suggest that "inadvertently" sorting of cargos and exporting proteins associated with senescence-repair by placental EVs may be associated with the dysfunction of placental development seen in missed miscarriage.

Placental extracellular vesicles retain biological activity after short-term storage (14 days) at 4 °C or room temperature.

INTRODUCTION: To investigate the role of placental extracellular vesicles (EVs), especially in pathological pregnancy, the use of freshly isolated EVs is often limited due to the sporadic and unpredictable availability of placental samples. Therefore, it is important to understand and use optimised storage conditions for placental EVs. In this study, we investigated different conditions for the short-term storage of placental micro- and nano-EVs and examined their biological activity. METHODS: Placental EVs were collected from first trimester placentae. EVs were suspended in PBS and aliquoted, and then stored for up to 14 days at room temperature, 4 °C or -20 °C. Total protein and DNA levels were measured at various time points. The ability of stored placental EVs to alter endothelial cell activation was quantified by monocyte adhesion assays. RESULTS: There was no difference in the concentration of placental micro- or nano-EVs between each time point, when stored at either room temperature or 4 °C. However, there was a significant loss of placental EVs after storage at -20 °C. There was no difference in protein or DNA levels of placental EVs when stored at either room temperature or 4 °C. Biological activity of placental EVs was retained for up to 14 days at either room temperature or 4 °C measured by monocyte adhesion assays. DISCUSSION: We have shown that placental micro- and nano-EVs are stable and retain biological activities following storage in PBS or media for 14 days at either room temperature or 4 °C.

Upregulation of pannexin-1 hemichannels explains the apparent death of the syncytiotrophoblast during human placental explant culture.

BACKGROUND: It has been reported that during the culture of human placental explants, the syncytiotrophoblast dies between 3 and 24 h and is then replaced within 48 h by a new syncytiotrophoblast layer formed by the fusion of underlying cytotrophoblasts. Most frequently the death of the syncytiotrophoblast is indicated by the uptake of nuclear stains such as propidium iodide (PI). This process is reportedly similar in both early and late gestation placental explants. METHODS: We cultured first trimester placental explants for up to 48 h and tested membrane intactness by exposure to PI. Connexin and pannexin mRNAs were quantified by RT-PCR and protein levels determined by immunofluorescence. The syncytiotrophoblast membrane leak was determined by culturing explants in the presence of hemichannel blockers. Extrusion of extracellular vesicles from the syncytiotrophoblast was quantified. RESULTS: Nuclei of the syncytiotrophoblast were stained with PI following approximately 4 h of culture and this was prevented by culturing the explants with pannexin-1 blockers. Expression of pannexin-1 hemichannels increased during explant culture (p = 0.0027). Extracellular vesicles were most abundantly extruded from the explants during the first 3 h of culture and the temporal pattern of extrusion was unaltered by blocking hemichannels. DISCUSSION: We show the mechanism of uptake of nuclear non-viability stains into the syncytiotrophoblast during explant culture is via upregulation of pannexin 1 hemichannels. Contrary to suggestions by some, the production of extracellular vesicles from cultured placental explants is not an in vitro artefact resulting from the apparent death of the syncytiotrophoblast in explant cultures.

Estimation of the burden of human placental micro- and nano-vesicles extruded into the maternal blood from 8 to 12 weeks of gestation.

BACKGROUND: The human placenta extrudes a variety of extracellular vesicles (EVs) into the maternal blood daily. These vesicles may be crucial to the adaptation of the maternal cardiovascular and immune systems to pregnancy. Quantifying the EVs that are released in early gestation is important to our understanding of how placental EVs may contribute to the regulation of maternal physiology. METHODS: EVs were isolated from first trimester placental explants and separated into micro- and nano-vesicles by differential centrifugation. The numbers of each type of EVs extruded from each milligram of placentae between gestational weeks 8 and 12 was determined by Nanoparticle Tracking Analysis. The total protein or DNA content of the vesicles was determined by BCA assay or Qubit® 2.0. RESULTS: Neither the number of micro- nor nano-EVs/mg explant (n = 49), nor the total protein (n = 19) and DNA content (n = 29) of these EVs changed significantly between 8 and 12 weeks of gestation. When the increasing placental weight with gestation was accounted for, the daily number of placental EVs extruded into the maternal blood increased by more than 100 fold between 8 and 12 weeks (micro-EVs 6.23 X 1014 and nano-EVs 1.84 X 1014 at 12 weeks, p = 0.0003). DISCUSSION: Constant production of micro- and nano-EVs per-milligram placenta, regardless of gestational age, and the increased daily burden of EVs across gestational age indicate these EVs have the potential to regulate maternal physiology from early pregnancy. Since total EV protein content, like EV numbers was, constant, this is a potentially reliable surrogate for quantifying EVs.

Karnofsky Performance Status Assessment: resident versus attending.

BACKGROUND: Karnofsky Performance Status (KPS) is a commonly used scale to assess a patient's functional status. METHODS: Between September 1999 and March 2000, 117 patients were independently evaluated and assigned KPS scores by both an attending physician and a resident physician at the time of radiation therapy simulation. RESULTS: Both attending and resident median assigned KPS score was 80. Attending and resident KPS scores were identical for 50 patients (43%). When KPS scores differed, this difference was of the smallest incremental value (10 points) in 50 patients (75%). The Pearson correlation coefficient is 0.85, significant at the 0.01 level. CONCLUSION: KPS scoring by radiation oncology attending physicians is similar to that by resident physicians.