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Objective To identify cervicovaginal fluid ( CVF) biomarkers predictive of spontaneous preterm birth in women with symptoms of preterm labour. Design Retrospective cohort study. Setting Melbourne, Australia. Population Women with a singleton pregnancy admitted to the Emergency Department between 22 and 36 weeks of gestation presenting with symptoms of preterm labour. Methods Two-dimensional electrophoresis was used to analyse the CVF proteome. Validation of putative biomarkers was performed using enzyme-linked immunosorbent assay ( ELISA) in an independent cohort. Optimal concentration thresholds of putative biomarkers were determined and the predictive efficacy for preterm birth was compared with that of fetal fibronectin. Main outcome measures Prediction of spontaneous preterm labour within 7 days. Results Differentially expressed proteins were identified by proteomic analysis in women presenting with 'threatened' preterm labour without cervical change who subsequently delivered preterm ( n = 12 women). ELISA validation using an independent cohort ( n = 129 women) found albumin and vitamin D-binding protein ( VDBP) to be significantly altered between women who subsequently experienced preterm birth and those who delivered at term. Prediction of preterm delivery within 7 days using a dual biomarker model (albumin/ VDBP) provided 66.7% sensitivity, 100% specificity, 100% positive predictive value ( PPV) and 96.7% negative predictive value ( NPV), compared with fetal fibronectin yielding 66.7, 87.9, 36.4 and 96.2%, respectively ( n = 64). Using the maximum number of screened samples, the predictive utility of albumin/ VDBP yielded a sensitivity of 77.8%, specificity and PPV of 100% and NPV of 98.0% ( n = 109). Conclusions The dual biomarker model of albumin/ VDBP is more efficacious than fetal fibronectin in predicting spontaneous preterm delivery in symptomatic women within 7 days. A clinical diagnostic trial is required to test this model on a larger population to confirm these findings and to further refine the predictive values. [ABSTRACT FROM AUTHOR]

OBJECTIVE: The purpose of this study was to investigate the temporal changes in immunoreactive cystatin A and the enzymatic activity of cathepsins B, H, L, and S in human cervicovaginal fluid (CVF) in late pregnancy and spontaneous labor. STUDY DESIGN: CVF was collected weekly (n = 95 women) from 36 weeks gestation until spontaneous term labor. Cystatin A was quantified using enzyme-linked immunosorbent assay. The enzyme activity of cathepsins B, H, L, and S was measured with fluorometric enzyme assay kits. RESULTS: Cystatin A significantly decreased towards (P = .016, 2-way analysis of variance) and during labor (P < .001, 2-way analysis of variance). Enzymatic activity of cathepsins B, H, and S did not change with labor onset (P = .452, P = .703, P = .411, respectively, 2-way analysis of variance). CONCLUSION: In late gestation, CVF-decreased expression of the cysteine protease inhibitor, cystatin A, is associated with labor. Although the role and contribution of cystatin A to increased extracellular matrix remodeling has yet to be elucidated, the data that were obtained are consistent with this hypothesis. [ABSTRACT FROM AUTHOR]

Reliably predicting spontaneous preterm birth remains challenging, therefore it persists as a major contributor to perinatal morbidity and mortality. The use of biomarkers to predict premature cervical shortening, a recognised risk factor for spontaneous preterm birth, is yet to be fully explored in current literature. This study evaluates seven cervicovaginal biochemical biomarkers as possible predictors of premature cervical shortening. Asymptomatic, high-risk women ( n  = 131) presenting to a specialised preterm birth prevention clinic were analysed through a retrospective data analysis. Cervicovaginal biochemical biomarker concentrations were obtained, and the shortest cervical length measurement, up to 28 weeks' gestation, was recorded. Associations between biomarker concentration and cervical length were then analysed. Of the seven biochemical biomarkers, Interleukin-1 Receptor Antagonist and Extracellular Matrix Protein-1 had statistically significant relationships with cervical shortening below 25 mm. Further investigation is required to validate these findings and any downstream clinical utility, with intentions to improve perinatal outcomes.IMPACT STATEMENT What is already known on this subject? Preterm birth is a major cause of perinatal morbidity and mortality. A woman's risk of delivering preterm is currently stratified using historical risk factors, mid-gestation cervical length, and biochemical biomarkers such as foetal fibronectin. What do the results of this study add? In a cohort of high-risk, asymptomatic pregnant women, two cervicovaginal biochemical biomarkers, Interleukin-1 Receptor Antagonist and Extracellular Matrix Protein-1, displayed associations with premature cervical shortening. What are the implications of these findings for clinical practice and/or further research? Further investigation into the possible clinical utility of these biochemical biomarkers is warranted, with a view to improving preterm birth prediction and antenatal resource utilisation, thereby reducing the burden of preterm birth and its sequelae in a cost-effective manner.

Introduction: In preeclampsia (PE), inadequate remodelling of spiral arterioles in the decidua basalis causes oxidative stress and subsequent increased release of antiangiogenic soluble endoglin (sENG) into the maternal circulation. Decidual mesenchymal stem/stromal cells (DMSCs) reside adjacent to endothelial cells in this vascular niche. Surprisingly, DMSCs express membrane-bound ENG (CD105). PE-affected DMSCs (PE-DMSCs) are abnormal and due to reduced extravillous invasion, more of them are present, but the significance of this is not known., Methods: DMSCs were isolated and characterised from normotensive control and severe-PE placentae. Extracellular vesicle (EV) types, shed microvesicles (sMV) and exosomes, were isolated from DMSC conditioned media (DMSC CM ), respectively. Secretion of ENG by DMSCs was assessed by ELISA of DMSC CM , with and without EV depletion. The effects of reducing ENG concentration, by blocking antibody, on human umbilical vein endothelial cell (HUVEC) attachment were assessed by xCELLigence real-time functional assays., Results: ENG was detected in DMSC CM and these levels significantly decreased when depleted of exosomes and sMV. There was no significant difference in the amount of ENG secreted by control DMSCs and PE-DMSCs. Blocking ENG in concentrated DMSC CM , used to treat HUVECs, improved endothelial cell attachment., Discussion: In normotensive pregnancies, DMSC secretion of ENG likely has a beneficial effect on endothelial cells. However, in PE pregnancies, shallow invasion of the spiral arterioles exposes more PE-DMSC derived sources of ENG (soluble and EV). The presence of these PE-DMSCs in the vascular niche contributes to endothelial cell dysfunction., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Preterm birth (PTB) is one of the most important problems that pose dilemmas for both the obstetrician and neonatologist, placing a heavy burden psychologically and financially on the families involved, and triggering high socio-economic costs to the public healthcare. The rate of PTB in Asian countries has been ranked at top globally. To reduce the PTB rate, to promote the prevention and intervention for PTB, and to better understand the pathophysiology underlying PTB, the Preterm Birth International Collaborative Australia branch (PREBIC-AA) was launched in 2017. A series scientific activities including organizing annual research symposiums has been planned and organized among Australasian countries. Here we briefly updated the current progress in clinical management and translational research on PTB in Australasian countries that have been participated in PREBIC-AA., (Copyright © 2022 Qiao, Menon, Ahn, Suzuki, Kshetrapal, Georgiou, Mesiano and Zhong.)

Preeclampsia (PE) is a serious medically important disorder of human pregnancy, which features de novo pregnancy-induced hypertension and proteinuria. The severe form of PE can progress to eclampsia, a convulsive, life-threatening condition. When placental growth and perfusion are abnormal, the placenta experiences oxidative stress and subsequently secretes abnormal amounts of certain pro-angiogenic factors (eg, PlGF) as well as anti-angiogenic factors (eg, sFlt-1) that enter the maternal circulation. The net effect is damage to the maternal vascular endothelium, which subsequently manifests as the clinical features of PE. Other than delivery of the fetus and placenta, curative treatments for PE have not yet been forthcoming, which reflects the complexity of the clinical syndrome. A major source of reactive oxygen species that contributes to the widespread maternal vascular endothelium damage is the PE-affected decidua. The role of decidua-derived mesenchymal stem/stromal cells (MSC) in normotensive and pathological placenta development is poorly understood. The ability to respond to an environment of oxidative damage is a "universal property" of MSC but the biological mechanisms that MSC employ in response to oxidative stress are compromised in PE. In this review, we discuss how MSC respond to oxidative stress in normotensive and pathological conditions. We also consider the possibility of manipulating the oxidative stress response of abnormal MSC as a therapeutic strategy to treat preeclampsia., (Copyright ©2022, Yale Journal of Biology and Medicine.)

Aims/hypothesis: Type 2 diabetes mellitus is a major cause of morbidity and death worldwide. Women with gestational diabetes mellitus (GDM) have greater than a sevenfold higher risk of developing type 2 diabetes in later life. Accurate methods for postpartum type 2 diabetes risk stratification are lacking. Circulating microRNAs (miRNAs) are well recognised as biomarkers/mediators of metabolic disease. We aimed to determine whether postpartum circulating miRNAs can predict the development of type 2 diabetes in women with previous GDM., Methods: In an observational study, plasma samples were collected at 12 weeks postpartum from 103 women following GDM pregnancy. Utilising a discovery approach, we measured 754 miRNAs in plasma from type 2 diabetes non-progressors (n = 11) and type 2 diabetes progressors (n = 10) using TaqMan-based real-time PCR on an OpenArray platform. Machine learning algorithms involving penalised logistic regression followed by bootstrapping were implemented., Results: Fifteen miRNAs were selected based on their importance in discriminating type 2 diabetes progressors from non-progressors in our discovery cohort. The levels of miRNA miR-369-3p remained significantly different (p < 0.05) between progressors and non-progressors in the validation sample set (n = 82; 71 non-progressors, 11 progressors) after adjusting for age and correcting for multiple comparisons. In a clinical model of prediction of type 2 diabetes that included six traditional risk factors (age, BMI, pregnancy fasting glucose, postpartum fasting glucose, cholesterol and triacylglycerols), the addition of the circulating miR-369-3p measured at 12 weeks postpartum improved the prediction of future type 2 diabetes from traditional AUC 0.83 (95% CI 0.68, 0.97) to an AUC 0.92 (95% CI 0.84, 1.00)., Conclusions: This is the first demonstration of miRNA-based type 2 diabetes prediction in women with previous GDM. Improved prediction will facilitate early lifestyle/drug intervention for type 2 diabetes prevention.

Introduction: The placenta is a short-lived organ, yet it shows signs of progressive ageing in the third trimester. Studies of ageing chorionic placental tissue have recently flourished, providing evidence of advanced ageing of tissues in the late/post-term (L/PT) period of gestation. However, ageing of the maternal aspect of the maternal-fetal interface, specifically the decidua basalis, is poorly understood. Here, we investigated whether the L/PT period was associated with advanced ageing and exhaustion of important decidua basalis mesenchymal stem/stromal cells (DMSCs) functions., Methods: In this study, DMSCs were isolated and characterised from early term (ET) and L/PT placental tissue and they were then investigated by employing various MSC potency and ageing assays. RNA sequencing was also performed to screen for specific microRNAs that are associated with stem cell exhaustion and ageing between ET- and L/PT-DMSCs., Results: L/PT-DMSCs, when compared to ET-DMSCs, showed significantly lower cell proliferation and a significant higher level of cell apoptosis. L/PT-DMSCs showed significantly lower resistance to oxidative stress and a significant decrease in antioxidant capacity compared with ET-DMSCs. Western blot analysis revealed increased expression of the stress-mediated P-p38MAPK protein in L/PT-DMSCs. RNA Sequencing showed microRNA (miR) miR-516b-5p, was present at significantly lower levels in L/PT-DMSCs. Inhibition of miR-516b-5p in ET-DMSCs revealed a decline in the ability of the inhibited cells to survive in extended cell culture., Discussion: These data provide the first evidence of advanced ageing and exhaustion of important stem cell functions in L/PT-DMSCs, and the involvement of specific miRs in the DMSC ageing process., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Despite sharing many common features, the relationship between ageing and parturition remains poorly understood. The decidua is a specialized lining of endometrial tissue, which develops in preparation for pregnancy. The structure and location of the decidua support its role as the physical scaffold for the growing embryo and placenta, and thus, it is vital to sustain pregnancy. Approaching term, the physical support properties of the decidua are naturally weakened to permit parturition. In this review, we hypothesize that the natural weakening of decidual tissue at parturition is promoted by the ageing process. Studies of the ageing-related functional and molecular changes in the decidua at parturition are reviewed and classified using hallmarks of ageing as the framework. The potential roles of decidual mesenchymal stem/stromal cell (DMSC) ageing in labor are also discussed because, although stem cell exhaustion is also a hallmark of ageing, its role in labor is not completely understood. In addition, the potential roles of extracellular vesicles secreted by DMSCs in labor, and their parturition-related miRNAs, are reviewed to gain further insight into this research area. In summary, the literature supports the notion that the decidua ages as the pregnancy progresses, and this may facilitate parturition, suggesting that ageing is the probable impetus of the gestational clocks in the decidua. This conceptual framework was developed to provide a better understanding of the natural ageing process of the decidua during parturition as well as to encourage future studies of the importance of healthy ageing for optimal pregnancy outcomes., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Oxidative stress and endothelial dysfunction contribute substantially to the pathogenesis of preeclampsia (PE). Decidual mesenchymal stem/stromal cells (DMSC), reportedly reduce endothelial cell dysfunction and alleviate PE-like symptoms in a murine model. However, as a therapeutic strategy, the use of whole DMSC presents significant technical limitations, which may be overcome by employing DMSC-secreted extracellular vesicles (DMSC&#95;EV). DMSC&#95;EV restoration of endothelial dysfunction through a paracrine effect may alleviate the clinical features of PE., Objective: To determine whether DMSC-secreted, extracellular vesicles (DMSC&#95;EV) restore endothelial cell function and reduce oxidative stress., Methods: DMSC were isolated from the placentae of uncomplicated term pregnancies and DMSC&#95;EV prepared by ultracentrifugation. Human umbilical vein endothelial cells (HUVEC) were treated with bacterial lipopolysaccharide (LPS), or with serum from PE patients, to model the effects of PE. DMSC&#95;EV were then added to treated HUVEC and their growth profiles, inflammatory state, and oxidative stress levels measured., Results: DMSC&#95;EV displayed characteristic features of extracellular vesicles. In both LPS- and PE serum-treatment models, addition of DMSC&#95;EV significantly increased HUVEC cell attachment and proliferation, and significantly reduced production of pro-inflammatory cytokine IL-6. The addition of DMSC&#95;EV to LPS-treated HUVEC had no significant effect on total antioxidant capacity, superoxide dismutase levels or on lipid peroxidation levels. In contrast, the addition of DMSC&#95;EV to PE serum-treated HUVEC resulted in a significant reduction in levels of lipid peroxidation., Conclusion: Addition of DMSC&#95;EV had beneficial effects in both LPS- and PE serum- treated HUVEC but the two treatment models to induce endothelial cell dysfunction showed differences. The LPS treatment of HUVEC model may not accurately model the endothelial cell dysfunction characteristic of PE. Human cell culture models of PE show that DMSC&#95;EV improve endothelial cell dysfunction in PE, but testing in in vivo models of PE is required., (Copyright © 2020 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All rights reserved.)

Ageing and parturition share common pathways, but their relationship remains poorly understood. Decidual cells undergo ageing as parturition approaches term, and these age-related changes may trigger labour. Mesenchymal stem/stromal cells (MSCs) are the predominant stem cell type in the decidua. Stem cell exhaustion is a hallmark of ageing, and thus ageing of decidual MSCs (DMSCs) may contribute to the functional changes in decidual tissue required for term spontaneous labour. Here, we determine whether DMSCs from patients undergoing spontaneous onset of labour (SOL-DMSCs) show evidence of ageing-related functional changes compared with those from patients not in labour (NIL-DMSCs), undergoing Caesarean section. Placentae were collected from term (37-40 weeks of gestation), SOL (n = 18) and NIL (n = 17) healthy patients. DMSCs were isolated from the decidua basalis that remained attached to the placenta after delivery. DMSCs displayed stem cell-like properties and were of maternal origin. Important cell properties and lipid profiles were assessed and compared between SOL- and NIL-DMSCs. SOL-DMSCs showed reduced proliferation and increased lipid peroxidation, migration, necrosis, mitochondrial apoptosis, IL-6 production and p38 MAPK levels compared with NIL-DMSCs (P < 0.05). SOL- and NIL-DMSCs also showed significant differences in lipid profiles in various phospholipids (phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine), sphingolipids (ceramide, sphingomyelin), triglycerides and acyl carnitine (P < 0.05). Overall, SOL-DMSCs had altered lipid profiles compared with NIL-DMSCs. In conclusion, SOL-DMSCs showed evidence of ageing-related reduced functionality, accumulation of cellular damage and changes in lipid profiles compared with NIL-DMSCs. These changes may be associated with term spontaneous labour., (© The Author(s) 2020. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Accurate prediction of spontaneous preterm labor/preterm birth in asymptomatic women remains an elusive clinical challenge because of the multi-etiological nature of preterm birth., Objective: The aim of this study was to develop and validate an immunoassay-based, multi-biomarker test to predict spontaneous preterm birth., Materials and Methods: This was an observational cohort study of women delivering from December 2017 to February 2019 at 2 maternity hospitals in Melbourne, Australia. Cervicovaginal fluid samples were collected from asymptomatic women at gestational week 16 +0 -24 +0 , and biomarker concentrations were quantified by enzyme-linked immunosorbent assay. Women were assigned to a training cohort (n = 136) and a validation cohort (n = 150) based on chronological delivery dates., Results: Seven candidate biomarkers representing key pathways in utero-cervical remodeling were discovered by high-throughput bioinformatic search, and their significance in both in vivo and in vitro studies was assessed. Using a combination of the biomarkers for the first 136 women allocated to the training cohort, we developed an algorithm to stratify term birth (n = 124) and spontaneous preterm birth (n = 12) samples with a sensitivity of 100% (95% confidence interval, 76-100%) and a specificity of 74% (95% confidence interval, 66-81%). The algorithm was further validated in a subsequent cohort of 150 women (n = 139 term birth and n = 11 preterm birth), achieving a sensitivity of 91% (95% confidence interval, 62-100%) and a specificity of 78% (95% confidence interval, 70-84%)., Conclusion: We have identified a panel of biomarkers that yield clinically useful diagnostic values when combined in a multiplex algorithm. The early identification of asymptomatic women at risk for preterm birth would allow women to be triaged to specialist clinics for further assessment and appropriate preventive treatment., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: We aimed to investigate the association of breastfeeding on postpartum glucose levels and lipid profiles in women diagnosed with gestational diabetes mellitus (GDM) and women without GDM., Methods: We performed a secondary analysis of a cohort study of 243 women, 159 women with GDM and 84 normally glucose tolerant women between 2012 and 2017. At approximately 6-10 weeks postpartum, we measured fasting blood glucose and plasma lipid levels. Breastfeeding behaviour was self-defined as exclusive breastfeeding or not exclusive breastfeeding., Results: The mean (SD) glucose in the group of women who breastfed exclusively was 4.6 (0.49) mmol/L, compared to 4.9 (0.58) mmol/L (95% CI 0.45, 0.15, p  <  0.001) among women who did not exclusively breastfeed. Among women with GDM, the reduction in fasting glucose in women who were breastfeeding was 0.22 mmol/L (95% CI 0.39, 0.05, p  = 0.004), and in women who were not GDM, the reduction was 0.14 mmol/L (95% CI 0.37, 0.09, p  = 0.24,). After adjustment for GDM status in pregnancy, maternal body mass index (BMI), maternal age and ethnicity, and exclusive breastfeeding was associated with a decreased fasting glucose of 0.19 (95% CI 0.318, 0.061, p  = 0.004). After similar adjustment, there was no significant difference in triglycerides, high density lipoprotein cholesterol or low-density lipoprotein cholesterol between women who were breastfeeding and women who were not breastfeeding., Conclusions: Breastfeeding is associated with a reduction in fasting glucose levels postpartum, but not maternal lipid profile. Breastfeeding may play a role in reducing glucose intolerance in women who have had GDM., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s). 2019.)

Background: Women with previous gestational diabetes mellitus (GDM) have evidence of postpartum β -cell dysfunction, which increases their risk of developing type 2 diabetes (T2DM) later in life. Elevated levels of circulating cell-free preproinsulin ( INS ) DNA correlate with dying β -cells in both mice and humans. The aim of this study was to determine if cell-free circulating INS DNA levels are higher in women with previous GDM who develop T2DM., Methods: We used droplet digital (dd) PCR to measure the levels of cell-free circulating methylated and unmethylated INS DNA in plasma from 97 women with normal glucose tolerance (NGT), 12 weeks following an index GDM pregnancy. Women were assessed for up to 10 years for the development of T2DM., Results: In the follow-up period, 22% of women developed T2DM. Compared with NGT women, total cell-free INS DNA levels were significantly higher in women who developed T2DM ( P = 0.02). There was no difference in cell-free circulating unmethylated and methylated INS DNA levels between NGT women and women who developed T2DM ( P = 0.09 and P = 0.07, respectively)., Conclusions: In women with a previous index GDM pregnancy, postpartum levels of cell-free circulating INS DNA are significantly higher in those women who later developed T2DM., Competing Interests: There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Aims: The risk of developing type 2 diabetes is greater in women with previous gestational diabetes mellitus (GDM). Apolipoprotein (Apo) species have been associated with the development of type 2 diabetes in the general population. The aim of this study was to determine if circulating levels of Apo species can predict development of type 2 diabetes in women with previous GDM., Methods: Apo AI, Apo AII, Apo B, Apo CII, Apo CIII and Apo E levels were measured in 95 women with normal glucose tolerance, 12 weeks following an index GDM pregnancy. Women were assessed for up to 10 years for the development of type 2 diabetes., Results: Postpartum Apo CIII levels, and Apo CIII/Apo AI, Apo CIII/Apo AII, Apo CIII/Apo CII, Apo CIII/Apo E and Apo E/Apo CIII ratios were significantly and positively associated with the development of type 2 diabetes. After controlling for age and BMI, these associations, except for the Apo E/Apo CIII ratio, remained significant. In a clinical model of prediction of type 2 diabetes that included age, BMI, and pregnancy and postnatal fasting glucose, the addition of Apo CIII levels, Apo CIII/Apo AI, Apo CIII/Apo AII, Apo CIII/Apo CII, and Apo CIII/Apo E resulted in a net reclassification improvement of 16.2%., Conclusions: High Apo CIII levels and the Apo CIII/Apo AI, Apo CIII/Apo AII, Apo CIII/Apo CII, and Apo CIII/Apo E ratios are all significant risk factors for the development of type 2 diabetes in women with a previous GDM pregnancy., Competing Interests: The authors declare that they have no conflict of interest., (© Georg Thieme Verlag KG Stuttgart · New York.)

Background: Maternal, cord blood and childhood adipokines have been associated with childhood obesity. We investigated whether postpartum maternal adipokines are associated with increased weight at 1 year of age in children of women with gestational diabetes (GDM)., Methods: Plasma leptin and adiponectin concentrations were measured in 160 women at approximately 12 weeks following pregnancy with GDM and compared with infant weight for length z-score at 1 year of age after adjustment for maternal and infant demographic variables., Results: No association was demonstrated between maternal postpartum leptin and adiponectin concentrations and infant weight for length z-score at 1 year of age.

Background: The placenta is an abundant source of mesenchymal stem/stromal cells (MSC), but our understanding of their functional properties remains limited. We previously created a placental-derived chorionic MSC (CMSC) cell line to overcome the difficulties associated with conducting extensive ex vivo optimization and experimental work on primary cells. The aim of this study was to characterize the migratory behavior of the CMSC29 cell line in vitro ., Methods: Stimulators of MSC migration, including two cytokines, stromal cell-derived factor-1α (SDF-1α) and hepatocyte growth factor (HGF), and a pharmacological agent, valproic acid (VPA), were tested for their ability to stimulate CMSC29 cell migration. Assessment of cell migration was performed using the xCELLigence Real-Time Cell Analyzer (RTCA)., Results: There was no significant increase in CMSC29 cell migration towards serum free medium with increasing concentration gradients of SDF-1α or HGF. In contrast, treating CMSC29 cells with VPA alone significantly increased their migration towards serum free medium., Conclusions: Immortalized CMSC29 cells retain important properties of primary CMSC, but their migratory properties are altered. CMSC29 cells do not migrate in response to factors that reportedly stimulate primary MSC/CMSC migration. However, CMSC29 increase their migration in response to VPA treatment alone. Further studies are needed to determine the mechanism by which VPA acts alone to stimulate CMSC29 migration. Still, this study provides evidence that VPA pre-treatment may improve the benefits of cell-based therapies that employ certain MSC sub-types., Competing Interests: Conflicts of Interest: The authors have no conflicts of interest to declare.

Background: To initiate tissue repair, mesenchymal stem/stromal cells (MSCs) must enter the blood stream, migrate to the targeted area, cross the endothelial barrier and home to the damaged tissue. This process is not yet fully understood in humans and thus, the aim of this study was to develop an ex vivo placental vessel perfusion method to examine human MSC movement from a blood vessel into human tissue. This will provide a better understanding of MSC migration, movement through the endothelial barrier and engraftment into target tissue, in a setting that more closely represents the in vivo state, compared with conventional in vitro human cell culture models. Moreover, important similarities and differences to animal experimental model systems may be revealed by this method., Methods: Human placental hTERT transformed MSC lines were labelled with live-cell fluorescence dyes, and then perfused into term human placental blood vessel. After labelled MSCs were perfused into the vessel, the vessel was dissected from the placenta and incubated at cell growth conditions. Following incubation, the vessel was washed thoroughly to remove unattached, labelled MSCs and then snap frozen for sectioning. After sectioning, immunofluorescence staining of the endothelium was carried out to detect if labelled MSCs crossed the endothelial barrier., Results: Twelve placental vessel perfusions were successfully completed. In eight of the twelve perfused vessels, qualitative assessment of immunofluorescence in sections (n=20, 5 µm sections/vessel) revealed labelled MSCs had crossed the endothelial barrier., Conclusions: The human placental ex vivo vessel perfusion method could be used to assess human MSC migration into human tissue. Cells of the MSC lines were able to adhere and transmigrate through the endothelial barrier in a manner similar to that of leukocytes. Notably, cells that transmigrated remained in close proximity to the endothelium, which is consistent with the reported MSC vascular niche in placental blood vessels., Competing Interests: Conflicts of Interest: The authors have no conflicts of interest to declare.

Preeclampsia (PE) is a hypertensive disorder of human pregnancy. Low-dose aspirin (acetylsalicylic acid) (60-150 mg/day) is used to prevent PE when taken early in pregnancy. The effect of aspirin on term PE remains uncertain. Abnormal placentation is a hallmark of PE and leads to increased placental oxidative stress, which triggers the release of anti-angiogenic factors that cause local damage to the decidual vasculature. The damage subsequently spreads systemically and culminates in maternal clinical symptoms. Decidua basalis mesenchymal stem/stromal cells (DMSCs) reside in a vascular microenvironment. In PE, DMSCs are exposed to abnormally high levels of oxidative stress and circulating inflammatory factors from the maternal blood. We previously showed that colony-forming unit ability and resistance to oxidative stress in DMSCs are reduced in MSCs derived from term PE pregnancies (PE-DMSCs). The action, if any, of aspirin on term PE-DMSCs has not been reported. In this study, aspirin (5 μg/mL) was found to significantly increase PE-DMSC adhesion compared to untreated PE-DMSCs and gestation-matched control DMSCs (p value < 0.001) but had no effect on PE-DMSC proliferation. ELISA analysis showed that aspirin significantly decreased the production of inflammatory cytokines IFN-γ (p value < 0.05) and IL-8 (p value < 0.001) in PE-DMSCs. In addition, aspirin treatment increased the antioxidant capacity of PE-DMSCs compared with the untreated group (p value < 0.05). This study is the first to reveal a novel, beneficial action of aspirin on PE-DMSCs from term PE pregnancies by improving their adhesion, suppressing their production of pro-inflammatory cytokines production, and increasing their antioxidant capacity. KEY MESSAGES: Preeclampsia (PE) is a serious hypertensive disorder of pregnancy. The risk of PE is reduced by aspirin but the mechanism is poorly understood. Decidua basalis mesenchymal stem/stromal cells (DMSCs) are abnormal in PE. Aspirin treatment improves multiple functions of PE-DMSCs. Improved DMSC function may contribute to the beneficial effect of aspirin.

Introduction: Chorionic mesenchymal stem/stromal cells (CMSC) can be isolated from the placenta in large numbers. Although their functions are yet to be fully elucidated, they have a role in tissue development and repair. To fulfil such a role, CMSC must be able to migrate to the microenvironment of the injury site. This process is not fully understood and the aim of this study therefore, was to examine in vitro CMSC migration in response to tissue inflammation and hypoxic conditioning., Methods: CMSC were derived from the chorionic villi. A trans-endothelium migration (TEM) assay was used to study CMSC migration through an activated endothelial cell monolayer using the HMEC-1 cell line. A cytokine array was used to identify and compare the cytokine production profile of activated versus non-activated HMEC-1., Results: There were significant changes in cytokine production by HMEC-1 cells following lipopolysaccharide (LPS) treatment and hypoxic conditioning. Despite this, results from the TEM assay showed no significant change in the average number of CMSC that migrated through the LPS activated HMEC-1 layer compared to the untreated control. Furthermore, there was no significant change in the average number of CMSC that migrated through the HMEC-1 monolayer when exposed to hypoxic (1% O 2 ), normoxic (8% O 2 ) or hyperoxic (21% O 2 ) conditions., Conclusion: These data suggest that cell functions such as transendothelial migration can vary between MSC derived from different tissues in response to the same biological cues., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Vitamin D-binding protein is a multifunctional serum protein with multiple actions related to normal health. Vitamin D-binding protein transports vitamin D and influences the metabolism of this key hormone but it also has additional immunomodulatory and actin-clearing properties. We investigated whether vitamin D-binding protein expression is altered in fetal growth restriction-associated placental dysfunction. Protein was extracted from 35 placentae derived from 17 healthy control subjects and 18 gestation-matched subjects with fetal growth restriction (FGR). FGR subjects were further subdivided as idiopathic ( n = 9) and nonidiopathic ( n = 9). Vitamin D-binding protein and 25(OH) vitamin D were measured by ELISA and normalized to protein concentration. The results showed significantly reduced levels of placental vitamin D-binding protein (control versus FGR, p < 0.05, Student's t -test) that were strongly associated with idiopathic fetal growth restriction ( p < 0.01, Kruskal-Wallis), whereas levels of vitamin D-binding protein were not associated with placental 25(OH) vitamin D stores ( p = 0.295, Pearson's correlation). As such, vitamin D-binding protein may be a factor in unexplained placental dysfunction associated with idiopathic fetal growth restriction and may potentially serve as a biomarker of this disease., Competing Interests: All the authors have no conflicting interests to declare.

Aims: Women with previous gestational diabetes mellitus (GDM) are at greater risk of developing type 2 diabetes. In the general population, the insulin-like growth factor (IGF) system has been implicated in the development of type 2 diabetes. The aim of this study was to determine if circulating IGF-I, IGF-II, IGFBP-1 and IGFBP-2 levels 12weeks following a GDM pregnancy are associated with an increased risk of developing type 2 diabetes., Methods: IGF-I, IGF-II, IGFBP-1 and IGFBP-2 levels were measured in 98 normal glucose tolerant women, 12weeks following an index GDM pregnancy using enzyme immunoassay. Women were assessed for up to 10years for the development of overt type 2 diabetes., Results: Among the 98 women with previous GDM, 21 (21%) developed diabetes during the median follow-up period of 8.5years. After adjusting for age and BMI, IGF-I and IGFBP-2 were significantly associated with the development of type 2 diabetes. In a clinical model of prediction of type 2 diabetes that included age, BMI, pregnancy fasting glucose and postnatal fasting glucose, the addition of IGF-I and IGFBP-2 resulted in an improvement in the net reclassification index of 17.8%., Conclusions: High postpartum IGF-I and low postpartum IGFBP-2 levels are a significant risk factor for the development of type 2 diabetes in women with a previous history of GDM. This is the first report that identifies IGF-I and IGFBP-2 as a potential biomarker for the prediction of type 2 diabetes in women with a history of GDM., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Background: Women with previous gestational diabetes mellitus (pGDM) are at increased risk of developing type 2 diabetes later in life. The aim of this study was to determine if circulating levels of metabolic hormones 12 weeks following a GDM pregnancy are associated with an increased risk of type 2 diabetes 8-10 years later., Methods: Fasting plasma concentrations of glucose, insulin, C-peptide, ghrelin, GIP, GLP-1, glucagon, leptin, PAI-1, resistin and visfatin were measured in 98 normal glucose tolerant women, 12 weeks following an index GDM pregnancy. Women were assessed every 2 years for up to 10 years for development of overt type 2 diabetes., Results: After a median follow-up period of 8.7 years, 22.5% of women with a pGDM pregnancy developed type 2 diabetes. Significant risk factors for the development of type 2 diabetes were fasting plasma glucose levels >5 mmol/L during pregnancy and at 12 weeks post-pregnancy. In addition, higher C-peptide levels and lower ghrelin levels at 12 weeks post-pregnancy were also significant risk factors for the development of type 2 diabetes., Conclusions: Fasting plasma glucose during pregnancy and post-partum, and post-partum C-peptide and ghrelin levels were significant risk factors for the development of type 2 diabetes in women with pGDM. This is the first report that identifies C-peptide and ghrelin as potential biomarkers for the prediction of type 2 diabetes in women with a history of GDM., (© 2014 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd.)

Aims/hypothesis: The risk of developing diabetes is greater for women with previous gestational diabetes mellitus (GDM). In the general population, plasma lipidomic analysis can identify individuals at risk of developing type 2 diabetes. The aim of this study was to determine whether circulating lipid levels 12 weeks following a GDM pregnancy were associated with an increased risk of developing type 2 diabetes., Methods: Plasma lipid profiles containing >300 lipids were measured in 104 normal glucose-tolerant women 12 weeks following an index GDM pregnancy using electrospray-ionisation tandem mass spectrometry. Women were assessed for 10 years for development of overt type 2 diabetes., Results: Among the 104 women with previous GDM, 21 (20%) developed diabetes during the median follow-up period of 8.5 years. Three lipid species, the cholesteryl ester species CE 20:4, the alkenylphosphatidylethanolamine species PE(P-36:2) and the phosphatidylserine species PS 38:4, were independently and positively associated with the development of type 2 diabetes. In a clinical model of prediction of type 2 diabetes that included age, BMI, and levels of pregnancy fasting glucose, postnatal fasting glucose, triacylglycerol and total cholesterol, the addition of these three lipid species resulted in an improvement in the net reclassification index of 22.3%., Conclusions/interpretation: The lipid species CE 20:4, PE(P-36:2) and PS 38:4 were significant risk factors for the development of type 2 diabetes in women with a previous history of GDM. This report is the first to use plasma lipidomic analysis to identify individual lipids as potential biomarkers for the prediction of type 2 diabetes in women with a history of GDM.

Preterm birth (PTB; birth before 37 completed weeks of gestation) remains the major cause of neonatal morbidity and mortality. The current generation of biomarkers predictive of PTB have limited utility. In pregnancy, the human cervicovaginal fluid (CVF) proteome is a reflection of the local biochemical milieu and is influenced by the physical changes occurring in the vagina, cervix and adjacent overlying fetal membranes. Term and preterm labor (PTL) share common pathways of cervical ripening, myometrial activation and fetal membranes rupture leading to birth. We therefore hypothesize that CVF biomarkers predictive of labor may be similar in both the term and preterm labor setting. In this review, we summarize some of the existing published literature as well as our team's breadth of work utilizing the CVF for the discovery and validation of putative CVF biomarkers predictive of human labor. Our team established an efficient method for collecting serial CVF samples for optimal 2-dimensional gel electrophoresis resolution and analysis. We first embarked on CVF biomarker discovery for the prediction of spontaneous onset of term labor using 2D-electrophoresis and solution array multiple analyte profiling. 2D-electrophoretic analyses were subsequently performed on CVF samples associated with PTB. Several proteins have been successfully validated and demonstrate that these biomarkers are associated with term and PTL and may be predictive of both term and PTL. In addition, the measurement of these putative biomarkers was found to be robust to the influences of vaginal microflora and/or semen. The future development of a multiple biomarker bed-side test would help improve the prediction of PTB and the clinical management of patients.

Objective: To identify cervicovaginal fluid (CVF) biomarkers predictive of spontaneous preterm birth in women with symptoms of preterm labour., Design: Retrospective cohort study., Setting: Melbourne, Australia., Population: Women with a singleton pregnancy admitted to the Emergency Department between 22 and 36 weeks of gestation presenting with symptoms of preterm labour., Methods: Two-dimensional electrophoresis was used to analyse the CVF proteome. Validation of putative biomarkers was performed using enzyme-linked immunosorbent assay (ELISA) in an independent cohort. Optimal concentration thresholds of putative biomarkers were determined and the predictive efficacy for preterm birth was compared with that of fetal fibronectin., Main Outcome Measures: Prediction of spontaneous preterm labour within 7 days., Results: Differentially expressed proteins were identified by proteomic analysis in women presenting with 'threatened' preterm labour without cervical change who subsequently delivered preterm (n = 12 women). ELISA validation using an independent cohort (n = 129 women) found albumin and vitamin D-binding protein (VDBP) to be significantly altered between women who subsequently experienced preterm birth and those who delivered at term. Prediction of preterm delivery within 7 days using a dual biomarker model (albumin/VDBP) provided 66.7% sensitivity, 100% specificity, 100% positive predictive value (PPV) and 96.7% negative predictive value (NPV), compared with fetal fibronectin yielding 66.7, 87.9, 36.4 and 96.2%, respectively (n = 64). Using the maximum number of screened samples, the predictive utility of albumin/VDBP yielded a sensitivity of 77.8%, specificity and PPV of 100% and NPV of 98.0% (n = 109)., Conclusions: The dual biomarker model of albumin/VDBP is more efficacious than fetal fibronectin in predicting spontaneous preterm delivery in symptomatic women within 7 days. A clinical diagnostic trial is required to test this model on a larger population to confirm these findings and to further refine the predictive values., (© 2014 Royal College of Obstetricians and Gynaecologists.)

Preterm labour and birth are a major cause of perinatal morbidity and mortality. Despite modern advances in obstetric and neonatal management, the rate of preterm birth in the developed world is increasing. Yet even though numerous risk factors associated with preterm birth have been identified, the ability to accurately predict when labour will occur remains elusive, whether it is at a term or preterm gestation. In the latter case, this is likely due to the multifactorial aetiology of preterm labour wherein women may display different clinical presentations that lead to preterm birth. The discovery of novel biomarkers that could reliably identify women who will subsequently deliver preterm may allow for timely medical intervention and targeted therapeutic treatments aimed at improving maternal and fetal outcomes. Various body fluids including amniotic fluid, urine, saliva, blood (serum/plasma), and cervicovaginal fluid all provide a rich protein source of putative biochemical markers that may be causative or reflective of the various pathophysiological disorders of pregnancy, including preterm labour. This short review will highlight recent advances in the field of biomarker discovery and the utility of single and multiple biomarkers for the prediction of preterm birth in the absence of intra-amniotic infection.

Introduction: Gestational diabetes mellitus (GDM) is characterised by maternal glucose intolerance and insulin resistance during pregnancy. Myostatin, initially identified as a negative regulator of muscle development may also function in the regulation of placental development and glucose uptake. Myostatin expression in placentae of GDM complicated pregnancies is unknown. However, higher myostatin levels occur in placentae of pregnancies complicated with preeclampsia. We hypothesise that myostatin will be differentially expressed in GDM complicated pregnancies., Methods: Myostatin concentrations (ELISA) were evaluated in plasma of presymptomatic women who later developed GDM and compared to plasma of normal glucose tolerant (NGT) women. Furthermore, myostatin protein expression (Western blot) was studied in placentae of pregnant women with GDM (treated with diet or insulin) compared to placentae of NGT women., Results: No significant difference in myostatin concentration was seen in plasma of pre-symptomatic GDM women compared to NGT women. In placenta significant differences in myostatin protein expressions (higher precursor; p < 0.05and lower dimer: p < 0.005) were observed in GDM complicated compared to NGT pregnancies. Furthermore, placentae of GDM women treated with insulin compared to diet have higher dimer (p < 0.005) and lower precursor (p < 0.05). Compared to lean women, placentae of obese NGT women were lower in myostatin dimer expression (p < 0.05)., Discussion: Myostatin expression in placental tissue is altered under stress conditions (e.g. obesity and abnormal glucose metabolism) found in pregnancies complicated with GDM. We hypothesise that myostatin is active in these placentae and could affect glucose homoeostasis and/or cytokine production thereby altering the function of the placenta., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

This work assessed the temporal coexpression of interleukin 1 (IL-1) and its inhibitor, IL-1 receptor antagonist (IL-1ra), in the cervicovaginal fluid (CVF) beyond 24 weeks gestation including women in spontaneous term labor. Two cohorts of women were recruited at 24 to 35 weeks' gestation (n = 65) and in late pregnancy (>36 weeks' gestation; n = 88). The CVF was serially collected either every 4 weeks between 24 and 35 weeks' gestation (n = 123 samples) or weekly during late pregnancy (n = 240 samples). The IL-1 and IL-1ra were quantitated by enzyme-linked immunosorbent assay, and the effect of vaginal microflora and unprotected sexual intercourse were also investigated. The IL-1β and IL-1ra remain unaltered between 24 and 35 weeks' gestation. At late pregnancy, IL-1α and β concentrations peak at 4 to 14 days prior to labor onset, while IL-1ra decreases with approaching spontaneous term labor (P < .05, 2-way analysis of variance). The IL-1 and IL-1ra were significantly correlated (P < .001, Pearson r). A combined biomarker model of IL-1α, IL-1β, and IL-1ra can predict term labor with 86% sensitivity and 92% specificity. This study indicates a shifting inflammatory balance in the gestational tissues prior to labor onset.

Vitamin D binding protein (VDBP) has previously been identified in the amniotic fluid and cervicovaginal fluid (CVF) of pregnant women. The biological functions of VDBP include acting as a carrier protein for vitamin D metabolites, the clearance of actin that is released during tissue injury and the augmentation of the pro-inflammatory response. This longitudinal observational study was conducted on 221 healthy pregnant women who spontaneously laboured and delivered either at term or preterm. Serial CVF samples were collected and VDBP was measured by ELISA. Binary logistic regression analysis was performed to assess the utility of VDBP as a predictor of labour. VDBP in the CVF did not change between 20 and 35 weeks' gestation. VDBP measured in-labour was significantly increased 4.2 to 7.4-fold compared to 4-7, 8-14 and 15-28 days before labour (P<0.05). VDBP concentration was 4.3-fold significantly higher at 0-3 days compared to 15-28 days pre-labour (P<0.05). The efficacy of VDBP to predict spontaneous labour onset within 3 days provided a positive and negative predictive value of 82.8% and 95.3% respectively (area under receiver operator characteristic curve  = 0.974). This longitudinal study of pregnant women suggests that VDBP in the CVF may be a useful predictor of labour.

The ability to recognise women who are at-risk of preterm labour (PTL) is often difficult. Over 50% of women who are identified with factors associated with an increased risk of preterm birth will ultimately deliver at term. The cervicovaginal fluid (CVF) comprises a range of proteins secreted by gestational tissues, making it an ideal candidate for the screening of differentially expressed proteins associated with PTL. CVF samples were collected from at-risk asymptomatic women. Two-dimensional gel electrophoresis techniques were used to examine the CVF proteome of women who spontaneously delivered preterm 11-22 days later compared with gestation-matched women who delivered at term. Five candidate biomarkers were selected for further validation in a larger independent cohort of asymptomatic women. Thioredoxin (TXN) and interleukin 1 receptor antagonist (IL1RN) concentrations in the CVF were found to be significantly reduced up to 90 days prior to spontaneous PTL compared with women who subsequently delivered at term. TXN was able to predict spontaneous PTL within 28 days after sampling with a high positive predictive value (PPV) and negative predictive value (NPV) of 75.0% and 96.4% respectively. IL1RN also showed comparable PPV and NPV of 72.7% and 95.7% respectively. The discovery of these differentially expressed proteins may assist in the development of a new predictive bedside test in identifying asymptomatic women who have an increased risk of spontaneous PTL.

A significant obstetric complication facing contemporary materno-fetal medicine is preterm premature rupture of the fetal membranes (preterm PROM), which occurs in 30% of all preterm births. The objective of this study was to identify differentially expressed proteins in the cervicovaginal fluid of asymptomatic women before the clinical manifestation of preterm PROM. The preterm PROM group comprised of women with samples collected 6-23 days before PROM, who subsequently delivered preterm (n=5). Women who spontaneously delivered at term served as gestation-matched controls (n=10). Two-dimensional difference in-gel electrophoresis was used to distinguish differential expression between the pooled groups and fold changes were subsequently confirmed by two-dimensional PAGE of individual samples. Spots of interest were identified by mass spectrometry. Proteins that were significantly reduced with impending preterm PROM included the following: thioredoxin (2.7-fold), interleukin 1 receptor antagonist (1.7-fold), fatty acid-binding protein 5 (2.1-fold), cystatin A (dimer; 1.9-fold), monocyte/neutrophil elastase inhibitor (1.6-fold), squamous cell carcinoma antigen-1 (2.1-fold) and γ-glutamyl cyclotransferase (3.0-fold). By contrast, annexin A3 (3.7-fold) and vitamin D binding protein (3.9-fold) were significantly increased with impending preterm PROM. Western blot analysis was also performed on an independent cohort of preterm PROM and control samples to validate these candidate biomarkers. These proteins have known biological functions in oxidative balance, anti-inflammatory activity, metabolism or protease inhibition that may facilitate membrane rupture.

Temporal expression of matrix metalloproteinase (MMP)-1, -2, -3, -7, -8, -9, -12, and -13, and tissue inhibitors of metalloproteinases (TIMPs)-1 and -2 in human cervicovaginal fluid (CVF) in term pregnancy and labor was investigated. Term parous women provided CVF samples that were grouped into labor, 1 to 3, 6 to 8, and 12 to 16 days before labor onset. Both MMPs and TIMPs (n = 60) were quantified using multiplex solution array and enzyme-linked immunosorbent assays, respectively. Further analysis of TIMP-1 (n = 180) was undertaken. All MMPs and TIMPs except MMP-12 and -13 were detected in the CVF. Matrix metalloproteinase 7, TIMP-1, and TIMP-2 were significantly increased in labor. Tissue inhibitors of metalloproteinase 1 was significantly increased up to 7 days before spontaneous labor onset. The data suggest a role of MMP-7 in the remodeling and rupture of fetal membranes and may reflect the homeostatic regulation of extracellular matrix remodeling of MMP-7 by TIMP-1 and TIMP-2.