Local vaginal bioelectrical impedance can predict preterm delivery in mice.

Preterm birth is a serious pregnancy complication that affects neonatal mortality, morbidity, and long-term neurological prognosis. Predicting spontaneous preterm delivery (PTD) is important for its management. While excluding the risk of PTD is important, identifying women at high risk of PTD is imperative for medical intervention. Currently used PTD prediction parameters in clinical practice have shown high negative predictive values, but low positive predictive values. We focused on sulfated and sialylated glycocalyx changes in the uterus and vagina prior to the onset of parturition and explored the potential of electrophysiological detection of these changes as a PTD prediction parameter with a high positive predictive value. In vivo local vaginal bioelectrical impedance (VZ) was measured using two different mouse PTD models. PTD was induced in ICR mice through the subcutaneous injection of mifepristone or local intrauterine injection of lipopolysaccharide (LPS). The PTD rates were 100% and 60% post-administration of mifepristone (16-20 h, n = 4) and LPS (12-24 h, n = 20), respectively. The local VZ values (15 and 10 h after mifepristone or LPS treatment, respectively) were significantly lower in the PTD group than in the non-PTD group. Receiver operator characteristic (ROC) curve analysis of VZ at 125 kHz as a predictor of PTD showed an area under the ROC curve of 1.00 and 0.77 and positive predictive values of 1.00 and 0.86, for the mifepristone and LPS models, respectively, suggesting that local VZ value can predict PTD. Histological examination of the LPS-treated model 6 h post-treatment revealed increased expression of sulfomucins and/or sulfated proteoglycans and sialomucins in the cervical epithelium, cervical stroma and vaginal stroma. In conclusion, local VZ values can determine sulfated and sialylated glycocalyx alterations within the uterus and vagina and might be a useful PTD prediction parameter.

A preliminary study on the glycosylation of the reproductive tract in the Ostrich (Struthio camelus camelus).

Glycosylation of the reproductive tract of an adult female red-necked ostrich (Struthio camelus camelus) carrying a fully formed calcified egg in her uterus when accidently killed by a blow to the head was examined using lectin histochemistry on samples from the infundibulum, magnum, uterus and vagina. Glycans in the luminal epithelium and underlying glands were described after staining with 23 lectins after neuraminidase pre-treatment in some cases. Ciliated and non-ciliated cells were evident at all levels in the luminal epithelium, the latter full of richly glycosylated secretory granules. The ciliated cells also showed glycosylation and, in the magnum, these cells often stained more intensely than the non-ciliated cells. High mannose and complex N-glycans, α1,6-linked fucosyl and sialic acid residues were present throughout the tract and there was a complete absence of GalNAcα1,3(LFucα1,2)Galß1,3/4GlcNAcß1- and rare terminal GalNAcα1- residues. Fucose in α1,2-linkage as H2 antigen and Ley was also rare in the luminal epithelium and completely absent in glands. Terminal galactose was present in the luminal epithelium apart from in the infundibulum. Gland epithelium showed similar glycosylation to the luminal epithelium except in the magnum where there were significant differences and here the glands were packed full of large secretory granules, unlike the glands in the rest of the tract. Each section of the tract had its own specific pattern of glycosylation which could be related to the stage of egg formation.

Increased Placental Cell Senescence and Oxidative Stress in Women with Pre-Eclampsia and Normotensive Post-Term Pregnancies.

Up to 11% of pregnancies extend to post-term with adverse obstetric events linked to pregnancies over 42 weeks. Oxidative stress and senescence (cells stop growing and dividing by irreversibly arresting their cell cycle and gradually ageing) can result in diminished cell function. There are no detailed studies of placental cell senescence markers across a range of gestational ages, although increased levels have been linked to pre-eclampsia before full term. This study aimed to determine placental senescence and oxidative markers across a range of gestational ages in women with uncomplicated pregnancies and those with a diagnosis of pre-eclampsia. Placentae were obtained from 37 women with uncomplicated pregnancies of 37-42 weeks and from 13 cases of pre-eclampsia of 31+2-41+2 weeks. The expression of markers of senescence, oxidative stress, and antioxidant defence (tumour suppressor protein p16INK4a, kinase inhibitor p21, interleukin-6 (IL-6), NADPH oxidase 4 (NOX4), glutathione peroxidases 1, 3, and 4 (GPx1, GPx3, and GPx4), placental growth factor (PlGF), and soluble fms-like tyrosine kinase-1 (sFlt-1)) genes was measured (quantitative real-time PCR). Protein abundance of p16INK4a, IL-6, NOX4, 8-hydroxy-2'-deoxy-guanosine (8-OHdG), and PlGF was assessed by immunocytochemistry. Placental NOX4 protein was higher in post-term than term deliveries and further increased by pre-eclampsia (p < 0.05 for all). P21 expression was higher in post-term placentae (p = 0.012) and in pre-eclampsia (p = 0.04), compared to term. Placental P16INK4a protein expression was increased post-term, compared to term (p = 0.01). In normotensive women, gestational age at delivery was negatively associated with GPx4 and PlGF (mRNA and protein) (p < 0.05 for all), whereas a positive correlation was seen with placental P21, NOX4, and P16INK4a (p < 0.05 for all) expression. Markers of placental oxidative stress and senescence appear to increase as gestational age increases, with antioxidant defences diminishing concomitantly. These observations increase our understanding of placental health and may contribute to assessment of the optimal gestational age for delivery.

Vaginal bioelectrical impedance determines uterine receptivity in mice.

STUDY QUESTION: Can vaginal bioelectrical impedance (VZ) electrophysiologically determine alterations of the endometrium in preparation for implantation? SUMMARY ANSWER: VZ can electrophysiologically detect the sulfation and sialylation changes in the uterine glycocalyx in preparation for implantation. WHAT IS KNOWN ALREADY: Uterine receptivity is associated with various glycosylation changes that affect negative charge density at the luminal epithelial cell surface. VZ has been used to monitor the oestrous cycle. STUDY DESIGN, SIZE, DURATION: Pathogen-free Jcl:ICR mice, aged 8-10 weeks, were used in this study. We conducted the following three steps to test our hypothesis that VZ may be used to determine uterine receptivity. First, to investigate whether VZ could determine alteration of sulfation and sialylation in the uterine glycocalyx, VZ was measured in mice with induced artificial sulfation and sialylation changes in the uterine glycocalyx (galactose-3-O-sulfotransferase 2 (GP3ST) + α(1,3/1,4) fucosyltransferase gene (FucT-III)-transferred group (n = 15) and in LacZ (encoding for ß-galactosidase)-transferred mice as a control group (n = 12)). Second, to investigate whether VZ could determine alterations of the endometrium in preparation for implantation, we measured VZ during the early stage of pregnancy (n = 12 each). Third, to investigate whether VZ could be used to evaluate uterine receptivity prospectively, VZ was measured in an implantation failure model mice. In 21 mice, local and transient suppression of signal transducer and activator of transcription-3 (Stat3) in the uterus were evaluated 1 day before implantation began, and 23 scramble decoy-transferred mice were used as a control group. PARTICIPANTS/MATERIALS, SETTING, METHODS: The VZ was measured at a frequency of 1 kHz in Jcl:ICR mice. Data were analysed using the Kruskal-Wallis test with Dunn's multiple comparisons, or the Student's t-test or Wilcoxon's rank-sum test with the Shapiro-Wilk normality test. The values of VZ were analysed using receiver operating characteristic (ROC) curve analysis to identify the optimal cut-off point to determine if this parameter predicted non-pregnancy. MAIN RESULTS AND THE ROLE OF CHANCE: Sulfation and sialylation changes induced in the luminal epithelial glycocalyx decreased the value of VZ. VZ showed a significant daily decrease during the early stage of pregnancy (Day 1.5 versus 2.5 p.c.: P < 0.005; Student's t-test, Day 2.5 versus 3.5 p.c.: P < 0.001; Wilcoxon's rank-sum test, Day 3.5 versus 4.5 p.c.: P < 0.005; Student's t-test, Day 4.5 versus 5.5 p.c.: P < 0.05; Student's t-test). One day before implantation began, VZ in the implantation failure model mice was significantly higher than in the control mice (P < 0.001, Wilcoxon's rank-sum test). The ROC curve analysis of VZ as a predictor of non-conception showed areas under the ROC curve of 0.91 (95% CI: 0.83-0.99). LIMITATIONS, REASONS FOR CAUTION: Although it is influenced by surface charge in the uterine epithelium, the mechanism whereby VZ changes during early pregnancy is still unexplained. WIDER IMPLICATIONS OF THE FINDINGS: Local bioelectrical impedance may help to prospectively evaluate uterine receptivity in women. Including the measurement of local bioelectrical impedance as part of a frozen-thawed embryo transfer strategy may improve the efficiency of ART. STUDY FUNDING/COMPETING INTEREST(S): This work was supported in part by the Japan Society for the Promotion of Science JSPS KAKENHI Grant (Nos. 19390429, 21390453, 16K11086 and 16K11087) from the Ministry of Education, Science and Culture of Japan (Tokyo, Japan) and Suzuken Memorial Foundation (Nagoya, Japan). The authors declare that they have no conflict of interest.

Placental endoplasmic reticulum stress in gestational diabetes: the potential for therapeutic intervention with chemical chaperones and antioxidants.

AIMS/HYPOTHESIS: The aim of this work was to determine whether placental endoplasmic reticulum (ER) stress may contribute to the pathophysiology of gestational diabetes mellitus (GDM) and to test the efficacy of chemical chaperones and antioxidant vitamins in ameliorating that stress in a trophoblast-like cell line in vitro. METHODS: Placental samples were obtained from women suffering from GDM and from normoglycaemic controls and were frozen immediately. Women with GDM had 2 h serum glucose levels > 9.0 mmol/l following a 75 g oral glucose tolerance test and were treated with diet and insulin when necessary. Western blotting was used to assess markers of ER stress. To test the effects of hyperglycaemia on the generation of ER stress, a new trophoblast-like cell line, BeWo-NG, was generated by culturing in a physiological glucose concentration of 5.5 mmol/l (over 20 passages) before challenging with 10 or 20 mmol/l glucose. RESULTS: All GDM patients were well-controlled (HbA1c 5.86 ± 0.55% or 40.64 ± 5.85 mmol/mol, n = 11). Low-grade ER stress was observed in the placental samples, with dilation of ER cisternae and increased phosphorylation of eukaryotic initiation factor 2 subunit α. Challenge of BeWo-NG with high glucose activated the same pathways, but this was as a result of acidosis of the culture medium rather than the glucose concentration per se. Addition of chemical chaperones 4-phenylbutyrate and tauroursodeoxycholic acid and vitamins C and E ameliorated the ER stress. CONCLUSIONS/INTERPRETATION: This is the first report of placental ER stress in GDM patients. Chemical chaperones and antioxidant vitamins represent potential therapeutic interventions for GDM.

The binucleate cell of okapi and giraffe placenta shows distinctive glycosylation compared with other ruminants: a lectin histochemical study.

The placenta of ruminants contains characteristic binucleate cells (BNC) with a highly conserved glycan structure which evolved early in Ruminant phylogenesis. Giraffe and Okapi placentae also contain these cells and it is not known whether they have a similar glycan array. We have used lectin histochemistry to examine the glycosylation of these cells in these species and compare them with bovine BNC which have a typical ruminant glycan composition. Two placentae, mid and near term, from Giraffe (Giraffa camelopardalis) and two term placenta of Okapi (Okapia johnstoni) were embedded in resin and stained with a panel of 23 lectins and compared with near-term bovine (Bos taurus) placenta. Significant differences were found in the glycans of Giraffe and Okapi BNC compared with those from the bovine, with little or no expression of terminal αN-acetylgalactosamine bound by Dolichos biflorus and Vicia villosa agglutinins which instead bound to placental blood vessels. Higher levels of N-acetylglucosamine bound by Lycopersicon esculentum and Phytolacca americana agglutinins were also apparent. Some differences between Okapi and Giraffe were evident. Most N-linked glycans were similarly expressed in all three species as were fucosyl residues. Interplacentomal areas in Giraffe and Bovine showed differences from the placentomal cells though no intercotyledonary BNC were apparent in Okapi. In conclusion, Giraffidae BNC developed different glycan biosynthetic pathways following their split from the Bovidae with further differences evolving as Okapi and Giraffe diverged from each other, affecting both inter and placentomal BNC which may have different functions during development.

Endotheliochorial placental glycosylation reflects evolutionary divergence between Felidae species (Felis catus and Panthera leo) and Canidae (Canisfamiliaris).

Endotheliochorial cat (Felis catus) and lion (Panthera leo) term placentae and one 6 week placenta (term 60-63 days) from a dog (Canis familiaris) were stained with a panel of 24 lectins to compare glycosylation at the feto-maternal interface. Glycan expression in lion and cat placentae was very similar apart from the occurrence of terminal α-galactose in the lion trophoblast. The dog differed in several respects, particularly in the trophoblast, consistent with species-specific glycotypes differing according to the degree of their evolutionary divergence. The data suggest that evolutionary effects on the glycotype are most readily observed in trophoblast.

Lectin histochemistry reveals two cytotrophoblast differentiation pathways during placental development in the feline (Feliscatus).

INTRODUCTION: Placental glycosylation has been examined on eight feline placentae ranging from approximately 15 to 60 days post-conception as little is known about changes in glycan distribution in this species. METHODS: Specimens were resin embedded and lectin histochemistry was applied to semi-thin sections using a panel of 24 lectins and an avidin-biotin revealing system. RESULTS: Abundant tri-tetraantennary complex N-glycan and α-galactosyl residues found in the syncytium in early pregnancy were greatly reduced in mid-pregnancy, though retained at the invasion front in the syncytium (N-glycan) or cytotrophoblast layer (αGal). Some other glycans were also uniquely present in invading cells. Abundant polylactosamine was found in the infolding basal lamina of syncytiotrophoblast and the apical villous cytotrophoblast membrane. Syncytial secretory granules often clustered near the apical membrane abutting maternal vessels. Decidual cells selectively expressed ß-galactosyl residues throughout pregnancy and highly branched N-glycan levels increased over time. DISCUSSION: Glycan distribution changes significantly over pregnancy, probably relating to the development of transport and invasive properties of trophoblast which in the endotheliochorial placenta reaches the level of the maternal vessels. Highly branched complex N-glycans, often associated with invasive cells, N-Acetylgalactosamine and terminal α-galactosyl residues are present at the invasion front abutting the junctional zone of the endometrium. Abundant polylactosamine on the syncytiotrophoblast basal lamina may reflect the presence of specialised adhesive interactions, while clustering of glycosylated granules apically is probably associated with secretion and absorption of material via maternal vasculature. It is suggested that lamellar and invasive cytotrophoblast represent distinct differentiation pathways. 246 words.

Perturbation of placental protein glycosylation by endoplasmic reticulum stress promotes maladaptation of maternal hepatic glucose metabolism.

Placental hormones orchestrate maternal metabolic adaptations to support pregnancy. We hypothesized that placental ER stress, which characterizes early-onset pre-eclampsia (ePE), compromises glycosylation, reducing hormone bioactivity and these maladaptations predispose the mother to metabolic disease in later life. We demonstrate ER stress reduces the complexity and sialylation of trophoblast protein N-glycosylation, while aberrant glycosylation of vascular endothelial growth factor reduced its bioactivity. ER stress alters the expression of 66 of the 146 genes annotated with "protein glycosylation" and reduces the expression of sialyltransferases. Using mouse placental explants, we show ER stress promotes the secretion of mis-glycosylated glycoproteins. Pregnant mice carrying placentas with junctional zone-specific ER stress have reduced blood glucose, anomalous hepatic glucose metabolism, increased cellular stress and elevated DNA methyltransferase 3A. Using pregnancy-specific glycoproteins as a readout, we also demonstrate aberrant glycosylation of placental proteins in women with ePE, thus providing a mechanistic link between ePE and subsequent maternal metabolic disorders.

Fucose, placental evolution and the glycocode.

A family of 13 fucosyltransferase genes has evolved to catalyze the addition of fucose in various linkage positions to nascent glycoproteins. Null mutations in mice are unearthing unsuspected functions for glycoprotein fucosylation that affect embryo implantation and growth of the conceptus. Furthermore, as we show here, histological studies demonstrate that a variety of fucosylated structures are found within the glycan-rich interface between trophectoderm and uterine epithelium. We suggest that conservation or change in fucosyltransferase gene expression over evolutionary time has played a role in determining the stability of the maternal-fetal interface and therefore in shaping reproductive compatibility and, in turn, speciation.

Heightened pro-inflammatory effect of preeclamptic placental microvesicles on peripheral blood immune cells in humans.

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.

Morphological and functional changes in placentas from prolonged pregnancies.

Prolonged pregnancy describes a pregnancy that progresses beyond 42 weeks' gestation (294 days). In humans, prolonged pregnancy is associated with increasing perinatal mortality, neonatal compromise and birth by Caesarean section. The underpinning reasons behind these increased risks are unknown; one potential explanation is reduced placental function due to ageing processes. This review describes the structural and functional changes seen in prolonged pregnancy in humans and in animal models. Prolonged pregnancies are associated with reduced placental growth, leading to an increase in fetal to placental weight ratio. Microscopic changes include aggregation of syncytiotrophoblast nuclei, reduced villous vascularity with a concomitant impairment of trophoblast transport processes (reduced pinocytosis); this is associated with increased evidence of oxidative stress, with downstream consequences including cellular senescence, autophagy and apoptosis; importantly many of these changes are similar to fetal growth restriction and pre-eclampsia. Thus, we argue that these observations provide evidence of ageing within the placenta, which may initially be adaptive but can become pathological leading to a reduction in placental function. This provides a biological basis for the increased risk of adverse outcomes observed in prolonged pregnancies. Greater insight into the effects and risks of placental ageing may be useful to guide clinicians on the management of prolonged pregnancies.

Observations on the glycosylation of the term placenta of the Indo-Pacific Bottlenose Dolphin (Tursiops aduncus): A lectin histochemical study.

INTRODUCTION: Little is known about the glycosylation of placental villi and areolae of cetaceans. Term tissue from the delivered placenta of an Indo-Pacific Bottlenose Dolphin (Tursiops aduncus) was examined using lectin histochemistry to compare trophoblast glycosylation in these two locations. METHODS: Placental blocks fixed in 10% formalin were resin-embedded before semithin sections were stained with 24 biotinylated lectins and an avidin-biotin revealing system. RESULTS: Areolar trophoblast was composed of large, bulbous cells packed with numerous granules compared to the smaller, cuboidal cells clothing the chorionic villi, which had a sparser, mainly subapical granule population. Both were richly glycosylated; generally areolar cells were more heavily stained apart from poor binding to some N-acetylgalactosamine and N-acetylglucosamine termini. Most striking was the distribution of α1,2-linked fucosyl residues, weakly expressed in villous trophoblast but intensely stained in some areolar cells, also terminal sialic acids. Some lectins bound in a variable fashion. Staining of terminal α-d-mannose, which locates mainly to lysosomes, was heavy in areolar cells compared to scattered irregular foci in villous cells. DISCUSSION AND CONCLUSION: The many intracellular inclusions reflect ongoing lysosomal breakdown of histotroph in areolar cells which often show heterogeneous glycosylation staining unlike the uniformly stained villous cells, possibly reflecting partial breakdown of ingested sialoglycoprotein, cell turnover or regional variation in uptake of histotroph. Our results indicate that Dolphin areolae are functionally distinct from villous trophoblast, performing absorptive and phagocytic functions similar to other Artiodactyla.

Mapping of CIP/KIP inhibitors, G1 cyclins D1, D3, E and p53 proteins in the rat term placenta.

As cell cycle regulation is fundamental to the normal growth and development of the placenta, the aim of the present study was to determine the immunolocalizations of cell cycle related proteins, which have key roles in proliferation, differentiation and apoptosis during the development of the rat placenta. Here immunohistochemistry has been used to localize G1 cyclins (D1, D3, E), which are major determinants of proliferation, CIP/KIP inhibitors (p21, p27, p57), p53 as a master regulator and proliferating cell nuclear antigen in all cell types of the rat term placenta. The proportion of each cell type immunolabeled was counted. Cyclin D1 and cyclin D3 were present mostly in cells of the fetal aspect of the placenta, whereas the G1/S cyclin E was present only in the spongio- and labyrinthine trophoblast populations. Among the CIP/KIP inhibitors, p21 was present only in cells of the fetal aspect whereas p27 and p57 were found in all cell types studied. p53 was only found in a small proportion of cells with no co-localization of p53 and p21. The data suggest that the cells of the fetal side of the rat placenta still have some proliferation potential which is kept in check by expression of the CIP/KIP cell cycle inhibitors, whereas cells of the maternal aspect have lost this potential. Apoptosis is only marginal in the term rat placenta. In conclusion, proliferation and apoptosis in rat placental cells appears controlled mostly by the CIP/KIP inhibitors in late pregnancy.

A new possible function for placental pericytes.

The pericyte is a multifunctional cell closely associated with endothelial cells and may play a role in angiogenesis and vessel stabilisation. Re-examination of over 1,100 micrographs from archival material used to investigate ultrastructural changes in placental development and pathology has identified previously undescribed structures associated with the pericyte of the human placental terminal villus. These structures take the form of outgrowths from the main body of the cell, with a narrow neck rich in cytoplasmic filaments, terminating in swollen tips which appear to bleb off the pericyte and form electron lucent stromal vesicles. Semi-quantitative analysis indicated that these features are present in some placentae from normal, term pregnancies but are increasingly found where capillaries show abnormalities such as a failure to form sinusoids, as in pregnancies complicated by diabetes, postmaturity, rhesus incompatibility and pre-eclampsia. This blebbing is compared with similar phenomena associated with apoptosis and zeiosis and it is suggested that it may contribute to fluid homeostasis where normal mechanisms are impaired by thickening or damage to endothelial cells.

Cell dynamics in human villous trophoblast.

BACKGROUND: Villous cytotrophoblast (vCTB) is a precursor cell population that supports the development of syncytiotrophoblast (vSTB), the high surface area barrier epithelium of the placental villus, and the primary interface between maternal and fetal tissue. In light of increasing evidence that the placenta can adapt to changing maternal environments or, under stress, can trigger maternal disease, we consider what properties of these cells empower them to exert a controlling influence on pregnancy progression and outcome. OBJECTIVE AND RATIONALE: How are cytotrophoblast proliferation and differentiation regulated in the human placental villus to allow for the increasing demands of the fetal and environmental challenges and stresses that may arise during pregnancy? SEARCH METHODS: PubMed was interrogated using relevant keywords and word roots combining trophoblast, villus/villous, syncytio/syncytium, placenta, stem, transcription factor (and the individual genes), signalling, apoptosis, autophagy (and the respective genes) from 1960 to the present. Since removal of trophoblast from its tissue environment is known to fundamentally change cell growth and differentiation kinetics, research that relied exclusively on cell culture has not been the main focus of this review, though it is mentioned where appropriate. Work on non-human placenta is not systematically covered, though mention is made where relevant hypotheses have emerged. OUTCOMES: The synthesis of data from the literature has led to a new hypothesis for vCTB dynamics. We propose that a reversible transition can occur from a reserve population in G0 to a mitotically active state. Cells from the in-cycle population can then differentiate irreversibly to intermediate cells that leave the cycle and turn on genes that confer the capacity to fuse with the overlying vSTB as well as other functions associated with syncytial barrier and transport function. We speculate that alterations in the rate of entry to the cell cycle, or return of cells in the mitotic fraction to G0, can occur in response to environmental challenge. We also review evidence on the life cycle of trophoblast from the time that fusion occurs, and point to gaps in knowledge of how large quantities of fetal DNA arrive in maternal circulation. We critique historical methodology and make a case for research to re-address questions about trophoblast lifecycle and dynamics in normal pregnancy and the common diseases of pre-eclampsia and fetal growth restriction, where altered trophoblast kinetics have long been postulated. WIDER IMPLICATIONS: The hypothesis requires experimental testing, moving research away from currently accepted methodology towards a new standard that includes representative cell and tissue sampling, assessment of cell cycle and differentiation parameters, and robust classification of cell subpopulations in villous trophoblast, with due attention to gestational age, maternal and fetal phenotype, disease and outcome.

A re-examination of the origins of placental bed giant cells.

In view of controversy about the source of placental multinuclear giant cells, we have re-examined the literature which clearly shows they are derived from trophoblastic elements that have populated the decidua. Archival material for electron microscopy from 17 to 18 week placentae demonstrates they can be found connected via desmosomes to the outer extravillous cytotrophoblast cells of anchoring columns, thus identifying a primary source. We suggest their formation is a terminal differentiation step occurring at all stages of invasion from the cell column to the myometrium, progressively reducing the invasive population.

Menstrual flow as a non-invasive source of endometrial organoids.

Assessment of the endometrium often necessitates a biopsy, which currently involves an invasive, transcervical procedure. Here, we present an alternative technique based on deriving organoids from menstrual flow. We demonstrate that organoids can be derived from gland fragments recovered from menstrual flow. To confirm they faithfully reflect the in vivo state we compared organoids derived from paired scratch biopsies and ensuing menstrual flow from patients undergoing in vitro fertilisation (IVF). We demonstrate that the two sets of organoids share the same transcriptome signature, derivation efficiency and proliferation rate. Furthermore, they respond similarly to sex steroids and early-pregnancy hormones, with changes in morphology, receptor expression, and production of 'uterine milk' proteins that mimic those during the late-secretory phase and early pregnancy. This technique has wide-ranging impact for non-invasive investigation and personalised approaches to treatment of common gynaecological conditions, such as endometriosis, and reproductive disorders, including failed implantation after IVF and recurrent miscarriage.

Evidence for the presence of air pollution nanoparticles in placental tissue cells.

Inhaled particulate matter (PM) from combustion- and friction-sourced air pollution adversely affects organs distant from the lung. A putative mechanism for the remote effect of inhaled PM is that ultrafine, nano-sized fraction (<100 nm) translocates across the air-tissue barrier, directly interacting with phagocytic tissue cells. Although PM is reported in other tissues, whether it is phagocytosed by non-respiratory tissue resident cells is unclear. Using the placenta as an accessible organ for phagocytic cells, we sought to seek evidence for air pollution-derived PM in tissue resident phagocytes. Macrophage-enriched placental cells (MEPCs) were isolated, and examined by light and electron microscopy. MEPC carbon was assessed by image analysis (mean µm2/1000 cells); particle composition and numbers were investigated using magnetic analyses and energy dispersive X-ray spectroscopy. MEPCs phagocytic capacity was assessed by culture with diesel exhaust PM in vitro. Fifteen placentas were analysed. Black inclusions morphologically compatible with inhaled PM were identified within MEPCs from all samples (mean ± SEM carbon loading, 1000 MEPCs/participant of 0.004 ± 0.001 µm2). High resolution scanning/transmission electron microscopy revealed abundant nano-sized particle aggregates within MEPCs. MEPC PM was predominantly carbonaceous but also co-associated with a range of trace metals, indicative of high temperature (i.e. exogenous) generation. MEPCs contained readily-measurable amounts of iron-rich, ferrimagnetic particles, in concentrations/particle number concentrations ranging, respectively, from 8 to 50 ng/g and 10 to 60.107 magnetic particles/g (wet wt) MEPCs. Extracted MEPCs (n = 20/ placenta) were phagocytic for PM since all cells showed increased carbon area after culture with diesel PM in vitro (mean ± SEM increase 7.55 ± 1.26 µm2 carbon PM). These findings demonstrate that inhaled, metal-bearing, air pollution-derived PM can not only translocate to distant organs, but is taken up by tissue resident phagocytes in vivo. The human placenta, and hence probably the fetus, thus appears to be a target for such particles.