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Introduction Premature ageing has been implicated in placental dysfunction. Senescence can be activated by oxidative stress, a key intermediary in the pathophysiology of pre-eclampsia. We examined senescence markers across normal gestation, and in pathological and post-mature pregnancies. Inducers of oxidative stress were used to mimic senescence changes in term explants. Methods Placental samples were collected with ethical approval and informed consent: first and second trimester samples from surgical terminations; term and pre-term controls, and early-onset pre-eclampsia samples from caesarean deliveries. Paraffin and EM blocks of post-mature placentas were from an archival collection. Term explants were subjected to hypoxia-reoxygenation (HR) or hydrogen peroxide (H2O2). Results p21 was increased significantly in term homogenates compared to first and second trimester samples, and was significantly higher in PE compared to term controls. Immunostaining revealed nuclear localisation of p21 and phosphorylated histone γH2AX in syncytiotrophoblast, with abundant foci in pathological and post-mature placentas. Abnormal nuclear appearances were observed in post-mature placentas. Sudan-Black-B staining demonstrated abundant lipofuscin, an aggregate of oxidised proteins, lipids and metals, in post-mature and pathological placentas. The percentage of nuclei positive for 8-hydroxy-2′-deoxy-guanosine, a marker of oxidised DNA/RNA, was increased in pathological placentas compared to age-matched controls. These changes could be mimicked by challenge with HR or H2O2. Discussion Senescence markers increase in normal placentas with gestational age, and are exaggerated in post-mature and pathological cases. Oxidative stress triggers equivalent changes in explants, and may precipitate senescence in vivo. The consequent pro-inflammatory senescence-associated secretory phenotype may contribute to the pathophysiology of pre-eclampsia., Highlights • Markers of senescence increase in healthy placentas with advancing gestational age. • Senescence changes are most extensive post-term, and in early-onset pre-eclampsia. • Oxidative stress can induce placental senescence in vitro. • Senescence changes are primarily restricted to the syncytiotrophoblast.

Background Preeclampsia (PE) is characterized by exaggerated apoptosis of the villous trophoblast of placental villi. Since p53 is a critical regulator of apoptosis we hypothesized that excessive apoptosis in PE is mediated by abnormal expression of proteins participating in the p53 pathway and that modulation of the p53 pathway alters trophoblast apoptosis in vitro. Methods Fresh placental villous tissue was collected from normal pregnancies and pregnancies complicated by PE; Western blotting and real-time PCR were performed on tissue lysate for protein and mRNA expression of p53 and downstream effector proteins, p21, Bax and caspases 3 and 8. To further assess the ability of p53 to modulate apoptosis within trophoblast, BeWo cells and placental villous tissue were exposed to the p53-activator, Nutlin-3, alone or in combination with the p53-inhibitor, Pifithrin-α (PFT- α). Equally, Mdm2 was knocked-down with siRNA. Results Protein expression of p53, p21 and Bax was significantly increased in pregnancies complicated by PE. Conversely, Mdm2 protein levels were significantly depleted in PE; immunohistochemistry showed these changes to be confined to trophoblast. Reduction in the negative feedback of p53 by Mdm2, using siRNA and Nutlin-3, caused an imbalance between p53 and Mdm2 that triggered apoptosis in term villous explants. In the case of Nutlin, this was attenuated by Pifithrin-α. Conclusions These data illustrate the potential for an imbalance in p53 and Mdm2 expression to promote excessive apoptosis in villous trophoblast. The upstream regulation of p53 and Mdm2, with regard to exaggerated apoptosis and autophagy in PE, merits further investigation.

Normal pregnancy is associated with the presence of circulating placental microvesicles (MVs). Increased MV shedding and altered immune activation are seen in patients with preeclampsia, suggesting that placental MVs may play a role in the pathophysiology of this disease. Therefore, the aim of this study was to investigate the activation of peripheral blood mononuclear cells (PBMCs) by MVs shed by first-trimester, normal term, and preeclamptic term placenta. First-trimester and preeclamptic term, but not normal term, placental-derived MVs activated PBMCs, as evidenced by elevated IL1B. Significant changes were also seen with several other cytokines and chemokines, and in general when compared to normal term MVs, preeclamptic MVs induced a greater pro-inflammatory response in PBMCs. Pretreatment of PBMCs with first-trimester or normal term placental MVs resulted in a dampened IL1B response to a subsequent lipopolysaccharide (LPS) challenge. In contrast, treatment of PBMCs with preeclamptic term placental MVs exacerbated the LPS response. This was also the case for several other cytokines and chemokines. These studies suggest that placental MVs can modulate basal peripheral immune cell activation and responsiveness to LPS during normal pregnancy, and that in preeclampsia this effect is exacerbated.

The hyena placenta is unique among carnivores in being hemochorial. It also has areas of erythrocyte uptake that differ from those seen in more commonly studied carnivores. The availability of timed midterm pregnancies made it possible to examine the organization of the placenta, the distribution of regions of columnar trophoblast and the nature of the heterophagous and hemophagous regions in well-preserved material. The labyrinth of the placenta is orderly arranged with periodic primary villi that have a surface of syncytial trophoblast. Secondary projections from the primary villi anastomose extensively. The junctional extreme of each primary villus forms an expanded tip covered by columnar trophoblast cells. Marginal to the hemochorial placenta, the paraplacenta is exposed to varying amounts of extravasated maternal blood which the cytotrophoblast cells ingest and destroy. Columnar trophoblast cells at the tips of the primary villi, in the villi of paraplacental margins of the labyrinth, in isolated patches within the labyrinth and beneath the allantochorionic plate are all capable of and are variably involved in erythrophagocytosis. Variations in the structure and the lectin histochemistry of the columnar trophoblast cells indicate variation depending on the exposure of these cells to erythrocytes and/or other histotrophic materials. The widespread distribution of the hemophagous regions suggests a nutritive function in addition to an iron transfer function of the columnar cytotrophoblast.

Genital ducts of three male Raja eglanteria were fixed and embedded in epoxy and methacrylate resin. Epoxy resin sections from the Leydig gland, upper and lower epididymis, ductus deferens and seminal vesicle were stained with 20 labelled lectins to examine their glycosylation. The Leydig gland consisted of columnar epithelial cells expressing N-linked glycans, N-acetyl galactosamine, glucosamine and lactosamine residues and sialic acid. Interspersed were ciliated cells of a different glycotype. The upper epididymis of cuboidal epithelium had a strongly glycosylated, ciliated apical surface and cytoplasmic granules that stained heavily with many lectins, with increased glycosylation compared to the Leydig gland. In the lower epididymis, tall, vacuolated cells showed some differences and a slight reduction in lectin staining. The ductus deferens contained two cell types and showed increased terminal N-acetyl galactosamine. The ciliated cuboidal epithelium of the seminal vesicle had marked differences from the ductus epithelium, with decreased N-acetyl galactosamine and lactosamine expression but increased subterminal N-acetyl lactosamine and galactosamine expression and sialylation. Spermatozoa were suspended in a glycosylated matrix and, in the seminal vesicle, were embedded in solid masses of matrix forming spermatozeugmata. These data show changes in glycan expression along the male genital tract, probably related to the nurture and maturation of the spermatozoa as they travel towards the seminal vesicle.