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Background: Nanotechnology involves the engineering of structures on a molecular level. Nanomedicine and nano-delivery systems have been designed to deliver therapeutic agents to a target site or organ in a controlled manner, maximizing efficacy while minimizing off-target effects of the therapeutic agent administered. In both reproductive medicine and obstetrics, developing innovative therapeutics is often tempered by fears of damage to the gamete, embryo or developing foetus or of negatively impacting a woman's reproductive potential. Thus, nanomedicine delivery systems may provide alternative targeted intervention strategies, treating the source of the disease and minimizing long-term consequences for the mother and/or her foetus.Objective and Rationale: This review summarizes the current state of nanomedicine technology in reproductive medicine and obstetrics, including safety, potential applications, future directions and the hurdles for translation.Search Methods: A comprehensive electronic literature search of PubMed and Web of Science databases was performed to identify studies published in English up until February 2020. Relevant keywords were used to obtain information regarding use of nanoparticle technology in fertility and gene therapy, early pregnancy complications (ectopic pregnancy and gestational trophoblastic disease) and obstetric complications (preeclampsia, foetal growth restriction, preterm birth and gestational diabetes) and for selective treatment of the mother or foetus. Safety of specific nanoparticles to the gamete, embryo and foetus was also investigated.Outcomes: Pre-clinical research in the development of nanoparticle therapeutic delivery is being undertaken in many fields of reproductive medicine. Non-hormonal-targeted nanoparticle therapy for fibroids and endometriosis may provide fertility-sparing medical management. Delivery of interventions via nanotechnology provides opportunities for gene manipulation and delivery in mammalian gametes. Targeting cytotoxic treatments to early pregnancy tissue provides an alternative approach to manage ectopic pregnancies and gestational trophoblastic disease. In pregnancy, nanotherapeutic delivery offers options to stably deliver silencing RNA and microRNA inhibitors to the placenta to regulate gene expression, opening doors to novel genetic treatments for preeclampsia and foetal growth restriction. Restricting delivery of teratogenic drugs to the maternal compartment (such as warfarin) may reduce risks to the foetus. Alternatively, targeted delivery of drugs to the foetus (such as those to treat foetal arrythmias) may minimize side effects for the mother.Wider Implications: We expect that further development of targeted therapies using nanoparticles in a reproductive setting has promise to eventually allow safe and directed treatments for conditions impacting the health and reproductive capacity of women and for the management of pregnancy and serious pregnancy complications. [ABSTRACT FROM AUTHOR]

Introduction: The discovery of new treatments that prevent or treat preeclampsia would be a major advance. Antiangiogenic factors soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sENG) are secreted in excess from the placenta, causing hypertension, endothelial dysfunction, and multiorgan injury. We recently identified metformin and esomeprazole as potential treatments for preeclampsia. Both reduce placental and endothelial secretion of sFlt-1 and soluble endoglin, and reduce endothelial dysfunction. Objectives: We set out to assess whether combining metformin and esomeprazole would additively reduce sFlt-1 and soluble endoglin secretion and reduce endothelial dysfunction (verses drug alone). Metformin and esomeprazole were added to primary placental cells and tissues, and endothelial cells and their effects on sFlt-1 and soluble endoglin secretion were assessed in vitro. Tumor necrosis factor-α (TNF-α) was added to endothelial cells to induce dysfunction in vitro. We examined the ability of metformin + esomeprazole to rescue TNF-α induced vascular cell adhesion molecule-1 (VCAM-1) and Endothelin-1 (ET-1) expression, leukocyte adhesion (markers of endothelial dysfunction). Results: Combining metformin and esomeprazole was additive at reducing sFlt-1 secretion and expression of sFlt-1 e15a mRNA isoform in primary cytotrophoblast, placental explants and endothelial cells. In contrast, no additive reduction in sENG was observed with combined metformin and esomeprazole. The low-dose combination of metformin + esomeprazole additively reduced TNF-α-induced VCAM-1 mRNA, but not VCAM-1 protein expression. There was no additive reduction when combining metformin and esomeprazole on TNF-α induced PBMC adhesion to endothelial cells. However, combining metformin and esomeprazole additively reduced ET-1 mRNA expression. Conclusions: In conclusion combining metformin and esomeprazole additively reduced secretion of sFlt-1, and markers of endothelial dysfunction. The combination of metformin and esomeprazole may provide a more effective treatment or prevention for preeclampsia compared to either as single agents. [ABSTRACT FROM AUTHOR]

Objective: sFLT-1 e15a is a recently described sFlt-1 variant that is placental and primate specific. As such, it may have potential as a biomarker. Using a newly developed ELISA, we measured maternal plasma sFLT-1 e15a levels in women with fetal growth restriction and pre-eclampsia.Method: We performed a nested case-control study where we measured total sFLT-1 and sFLT-1 e15a plasma protein concentrations. Samples, selected from a prospective cohort study, consisted of 87 healthy controls, 11 cases that developed term preeclampsia and 20 cases where there was fetal growth restriction. We also measured sFLT-1 and sFLT-1 e15a plasma concentrations in a separate cohort: 15 cases of preterm preeclampsia and 24 healthy controls.Results: The prospective case-control cohort demonstrated significantly increased sFLT-1 e15a among cases with term fetal growth restriction (p < 0.05). We also observed that total sFLT-1 (this ELISA indiscriminately detects all variants) was significantly increased in term preeclampsia (p < 0.0001), but not fetal growth restriction. The separate cohort of early-onset preeclamptics showed significantly increased sFLT-1 e15a levels (p < 0.0001).Conclusion: Plasma sFLT-1 e15a is significantly increased in early-onset preeclampsia and term fetal growth restriction. Further assessment of the benefit for sFLT-1 e15a testing in prediction or diagnosis of these disease states is warranted. [ABSTRACT FROM AUTHOR]

Preeclampsia is a heterogeneous and multiorgan cardiovascular disorder of pregnancy. Here, we report the development of a novel strip‐based lateral flow assay (LFA) using lanthanide‐doped upconversion nanoparticles conjugated to antibodies targeting two different biomarkers for detection of preeclampsia. We first measured circulating plasma FKBPL and CD44 protein concentrations from individuals with early‐onset preeclampsia (EOPE), using ELISA. We confirmed that the CD44/FKBPL ratio is reduced in EOPE with a good diagnostic potential. Using our rapid LFA prototypes, we achieved an improved lower limit of detection: 10 pg ml−1 for FKBPL and 15 pg ml−1 for CD44, which is more than one order lower than the standard ELISA method. Using clinical samples, a cut‐off value of 1.24 for CD44/FKBPL ratio provided positive predictive value of 100 % and the negative predictive value of 91 %. Our LFA shows promise as a rapid and highly sensitive point‐of‐care test for preeclampsia. [ABSTRACT FROM AUTHOR]

Preeclampsia is a heterogeneous and multiorgan cardiovascular disorder of pregnancy. Here, we report the development of a novel strip‐based lateral flow assay (LFA) using lanthanide‐doped upconversion nanoparticles conjugated to antibodies targeting two different biomarkers for detection of preeclampsia. We first measured circulating plasma FKBPL and CD44 protein concentrations from individuals with early‐onset preeclampsia (EOPE), using ELISA. We confirmed that the CD44/FKBPL ratio is reduced in EOPE with a good diagnostic potential. Using our rapid LFA prototypes, we achieved an improved lower limit of detection: 10 pg ml−1 for FKBPL and 15 pg ml−1 for CD44, which is more than one order lower than the standard ELISA method. Using clinical samples, a cut‐off value of 1.24 for CD44/FKBPL ratio provided positive predictive value of 100 % and the negative predictive value of 91 %. Our LFA shows promise as a rapid and highly sensitive point‐of‐care test for preeclampsia. [ABSTRACT FROM AUTHOR]

Background: It is not known whether fasting affects levels of circulating placenta-specific transcripts.Objective: To assess whether a glucose load affects circulating placenta-specific transcripts.Method: RNA was extracted from paired blood samples (fasting and 1-h post 75 g oral glucose) from 22 women. Placenta-specific genes were measured by RT-qPCR.Results: There was no change in ADM, CSH1, PAPPA2, PSG1 or TAC3 expression between fasting and post-glucose states. However, HTRA1 decreased after glucose load.Conclusion: Maternal fasting state does not influence expression of the majority of placenta-specific genes but may need to be accounted for when validating biomarkers of placental disease. [ABSTRACT FROM AUTHOR]

STUDY QUESTION What is the nature of cellular Corin expression in human gestational tissues? SUMMARY ANSWER CORIN is expressed in non-pregnant late secretory phase endometrium, first trimester human implantation sites and is up-regulated with decidualization ex vivo. WHAT IS KNOWN ALREADY Adequate trophoblast invasion and spiral artery remodeling/transformation is critical for successful implantation. CORIN, best known for its role in activating atrial natruietic peptide (ANP) to regulate blood pressure, has recently been proposed to be centrally involved in trophoblast invasion and spiral artery remodeling. It is postulated that ANP, activated by CORIN, promotes trophoblast invasion and that a deficiency causes pre-eclampsia. Mice deficient in either Corin or ANP displayed poor trophoblast invasion, impaired spiral artery remodeling and phenocopied human pre-eclampsia. However, the precise cellular localization of CORIN within human gestational tissues has not been well characterized. STUDY DESIGN, SIZE, DURATION We measured CORIN protein localization in a number of human gestational tissues relevant to early embryo/placental implantation: non-pregnant (NP) endometrial biopsies (n = 5 per phase of the menstrual cycle), first trimester placental bed biopsies (n = 12) and pre-term control (n = 10) and severe early onset preeclamptic placentas (n = 15). Endometrial stromal cells were isolated from human endometrial biopsies (n = 5) and induced to decidualize ex vivo. Finally, CORIN concentrations were measured in serum obtained from pregnant women during the first trimester of whom, 56 subsequently ended up with a healthy term delivery (controls), 18 developed fetal growth restriction (FGR) and 21 had a miscarriage. PARTICIPANTS/MATERIALS, SETTING, METHODS We performed immunohistochemistry to assess CORIN localization. Changes in Corin mRNA expression in human endometrial stromal cells decidualized ex vivo were measured by quantitative RT–PCR, and levels of CORIN within human sera were measured by ELISA. MAIN RESULTS AND THE ROLE OF CHANCE CORIN was expressed in both NP late secretory phase endometrium and first trimester decidua within placental bed biopsies. Importantly, decidualization of primary human endometrial cells ex vivo significantly increased Corin expression (P < 0.05). CORIN was also detected within the villous cytotrophoblast, but there was no change in mRNA levels in placentas complicated by severe preterm pre-eclampsia when compared with pre-term controls. Although CORIN was detected in first trimester serum, levels did not change across gestation, nor could they predict miscarriage or FGR (other disorders of impaired placental invasion). LIMITATIONS, REASONS FOR CAUTION Owing to the fact that we utilized early pregnancy human specimens, this is mainly a descriptive study with a limited amount of functional experiments. WIDER IMPLICATIONS OF THE FINDINGS This is the first study to thoroughly characterize Corin mRNA and protein expression in human gestational tissue. Our findings support recent data from murine studies collectively suggesting that CORIN plays a critical role in trophoblast migration and spiral artery remodeling during early pregnancy in humans. Therefore, further studies of CORIN biology in early pregnancy may identify new therapeutic targets to improve implantation quality in early pregnancy and potentially reduce the rates of pregnancy complications caused by inadequate implantation (pre-eclampsia, FGR and miscarriage). STUDY FUNDING/COMPETING INTEREST(S) This study was supported by The National Health and Medical Research Council of Australia (Salary support #490970, #490995). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the ... [ABSTRACT FROM PUBLISHER]

Abstract: A shift from type 1 to type 2 immune bias is essential for successful implantation and pregnancy. ST2 is a receptor present on type 2 immune cells and IL-33 was recently discovered to be its ligand. We measured serum levels of soluble ST2 (sST2) and IL-33 across the mid-first trimester and examined whether levels were dysregulated among women with a viable fetus but destined to miscarry. While there was no significant change in sST2 or IL-3 across the first trimester, we found that women with live pregnancies destined to fail had dysregulated levels of serum IL-33, and potentially sST2 at six weeks’ gestation. [Copyright &y& Elsevier]

Preeclampsia is a major pregnancy complication, characterized by severe endothelial dysfunction, hypertension and maternal end-organ damage. Soluble endoglin is an anti-angiogenic protein released from placenta and thought to play a central role in causing the endothelial dysfunction and maternal organ injury seen in severe preeclampsia. We recently reported MMP-14 was the protease producing placentally-derived soluble endoglin by cleaving full-length endoglin present on the syncytiotrophoblast surface. This find identifies a specific drug target for severe preeclampsia; interfering with MMP- 14 mediated cleavage of endoglin could decrease soluble endoglin production, ameliorating clinical disease. However, experimental MMP-14 inhibition alone only partially repressed soluble endoglin production, implying other proteases might have a role in producing soluble endoglin. Here we investigated whether MMP-15-phylogenetically the closest MMP relative to MMP-14 with 66% sequence similarity-also cleaves endoglin to produce soluble endoglin. MMP-15 was localized to the syncytiotrophoblast layer of the placenta, the same site where endoglin was localized. Interestingly, it was significantly (p = 0.03) up-regulated in placentas from severe early-onset preeclamptic pregnancies (n = 8) compared to gestationally matched preterm controls (n = 8). However, siRNA knockdown of MMP-15 yielded no significant decrease of soluble endoglin production from either HUVECs or syncytialised BeWo cells in vitro. Importantly, concurrent siRNA knockdown of both MMP-14 and MMP-15 in HUVECS did not yield further decrease in soluble endoglin production compared to MMP-14 siRNA alone. We conclude MMP-15 is up-regulated in preeclampsia, but does not cleave endoglin to produce soluble endoglin. [ABSTRACT FROM AUTHOR]

Miscarriage is the most common complication of pregnancy. Pre-clinical miscarriage has an estimated incidence of 30%, whilst clinical miscarriage has an incidence of 12-15%. Two thirds of pregnancies lost to miscarriage are believed to be attributable to defective placentation, thus a number of studies have sought to identify markers of defective placentation that could be used as clinical biomarkers of miscarriage. Decreased soluble FMS-like tyrosine kinase-1 (sFlt1), placental growth factor (PlGF), and soluble endoglin (sEng) in the maternal circulation during the first trimester have recently been proposed as potential markers of pregnancy loss. However, in these studies clinical samples were only obtained once women had presented with symptoms of miscarriage. In this study we prospectively screened serum samples collected from asymptomatic women with a viable fetus. We assessed maternal serum levels of sFlt1, PlGF and sEng across the first trimester of normal pregnancy and compared levels between women who continued to a live birth, to those who subsequently miscarried. Both sFlt1 and PlGF significantly (p<0.05) increased across gestation in normal pregnancy with serum levels rising from 0.65±0.12 ng/ml at 6 weeks to 1.85±0.24 ng/ml at 12 weeks for sFlt1, and 57.2±19.2 pg/ml to 106±22.7 pg/ml for PlGF. sEng remained unchanged throughout the the first trimester. Importantly we detected a significant (35%, p<0.05) decrease in sFlt1 levels between our control and miscarriage cohort, however there was significant overlap between cases and controls, suggesting serum sFlt1 is unlikely to be useful as a clinical biomarker in asymptomatic women. Nevertheless, our data suggests a dysregulation of angiogenic factors may be involved in the pathophysiology of miscarriage. [ABSTRACT FROM AUTHOR]

Candidate stem/progenitor cells have been identified in mouse endometrium as label-retaining cells (LRCs). The role of endometrial stem/progenitor cells in initiating estrogen-stimulated endometrial growth in prepubertal and cycling mice was investigated following a single 17β-estradiol (E2) injection in bromodeoxyuridine (BrdU)-labeled and -chased (LRC), ovariectomised mice. Proliferating (BrdU+/Ki-67+) and mitotic (BrdU+/PH3+) epithelial LRCs were first detected in prepubertal mice 8 hours following E2 treatment, initiating the proliferative response. In contrast, all epithelial LRCs and 16% of epithelial cells in cycling mice proliferated within 2 hours. In cycling mice, 12% of stromal LRCs initiated a proliferative response 8 hours after E2. Proliferating epithelial LRCs and most stromal LRCs (85%) lacked estrogen receptor-α (ESR1). These findings suggest that endometrial epithelial LRCs function as stem/progenitor cells by receiving proliferative signals from neighboring ESR1+ niche cells to initiate the growth of the epithelium during development, while mature epithelial cells may undergo self-replication in cycling endometrium. [ABSTRACT FROM PUBLISHER]

The contribution of the high abundance of inflammatory cells present in the human endometrium prior to and during menstruation is unknown with respect to endometrial repair and/or menstruation. In this study, the presence and localisation of markers for key inflammatory cells have been examined in a mouse model of endometrial breakdown and repair and the functional contribution of neutrophils has been determined. In the model, decidualisation is artificially induced and progesterone support withdrawn; the endometrial tissue progressively breaks down by 24 h after progesterone withdrawal and, by 48 h, has usually undergone complete repair. Neutrophils have been identified in low abundance in decidual tissue, rise in number during breakdown and are most abundant during early repair. Macrophages are barely detectable during breakdown or repair in this model, whereas uterine natural killer cells are found only in intact decidua. The functional contribution of neutrophils to endometrial breakdown and repair has been assessed via neutrophil depletion by using the antibody RB6-8C5. This antibody significantly depletes neutrophils from the circulation and tissue, affects endometrial breakdown and markedly delays endometrial repair. This study has therefore demonstrated that neutrophils are the most abundant leucocyte in this model and that they play an important functional role in the processes of endometrial breakdown and repair. [ABSTRACT FROM AUTHOR]

Objective: Fetal macrosomia is a major risk factor for shoulder dystocia, which can lead to birth asphyxia, maternal and neonatal traumatic injuries, and perinatal death. If macrosomia is diagnosed in the antenatal period, labour can be induced to decrease shoulder dystocia. But current clinical methods to diagnose fetal macrosomia antenatally perform with poor accuracy. Therefore, improved methods to accurately diagnose fetal macrosomia are required. Blood biomarkers that predict fetal macrosomia could be one such novel diagnostic strategy. We undertook a nested case–control study from a prospective collection of 1000 blood samples collected at 36 weeks' gestation. We analysed plasma samples from 52 women who subsequently delivered a macrosomic (> 95th centile for gestational age) infant and 106 controls. Circulating concentrations of the proteins COBLL1, CSH1, HSD3B1, EGFL6, XAGE3, S100P, PAPPA-1, ERBB2 were assessed for their ability to predict macrosomic infants. Results: We did not identify any significant changes in the plasma concentrations of COBLL1, CSH1, HSD3B1, EGFL6, XAGE3, S100P, PAPPA-1, ERBB2 from women who subsequently delivered macrosomic neonates relative to control samples. Although we have not identified any potential biomarkers of fetal macrosomia, we have ruled out these particular eight protein candidates. [ABSTRACT FROM AUTHOR]

Background: Preeclampsia is a severe complication of pregnancy which is attributed to placental dysfunction. The retrotransposon, Paternal Expressed Gene 10 (PEG10) harbours critical placental functions pertaining to placental trophoblast cells. Limited evidence exists on whether PEG10 is involved in preeclampsia pathogenesis. This study characterised the expression and regulation of PEG10 in placentas from patients with early-onset preeclampsia compared to gestation-matched controls. Methods: PEG10 expression was measured in plasma and placentas collected from patients with early-onset preeclampsia (< 34 weeks') and gestation-matched controls using ELISA (protein) and RT-qPCR (mRNA). First-trimester human trophoblast stem cells (hTSCs) were used for in vitro studies. PEG10 expression was measured during hTSC differentiation and hTSC exposure to hypoxia (1% O2) and inflammatory cytokines (IL-6 and TNFα) using RT-qPCR. Functional studies used PEG10 siRNA to measure the effect of reduced PEG10 on canonical TGF- β signalling and proliferation using luciferase and xCELLigence assays, respectively. Results: PEG10 mRNA expression was significantly reduced in placentas from patients with early-onset preeclampsia (< 34 weeks' gestation) relative to controls (p = 0.04, n = 78 vs n = 18 controls). PEG10 protein expression was also reduced in preeclamptic placentas (p = 0.03, n = 5 vs n = 5 controls, blinded assessment of immunohistochemical staining), but neither PEG10 mRNA nor protein could be detected in maternal circulation. PEG10 was most highly expressed in hTSCs, and its expression was reduced as hTSCs differentiated into syncytiotrophoblasts (p < 0.0001) and extravillous trophoblasts (p < 0.001). Trophoblast differentiation was not altered when hTSCs were treated with PEG10 siRNA (n = 5 vs n = 5 controls). PEG10 was significantly reduced in hTSCs exposed to hypoxia (p < 0.01). PEG10 was also reduced in hTSCs treated with the inflammatory cytokine TNF α (p < 0.01), but not IL-6. PEG10 knocked down (siRNA) in hTSCs showed reduced activation of the canonical TGF-β signalling effector, the SMAD binding element (p < 0.05) relative to controls. PEG10 knockdown in hTSCs however was not associated with any significant alterations in proliferation. Conclusions: Placental PEG10 is reduced in patients with early-onset preeclampsia. In vitro studies suggest that hypoxia and inflammation may contribute to PEG10 downregulation. Reduced PEG10 alters canonical TGF- β signalling, and thus may be involved in trophoblast dysfunction associated with this pathway. [ABSTRACT FROM AUTHOR]

Spontaneous preterm birth is the leading cause of perinatal morbidity and mortality. Tocolytics are drugs used in cases of imminent preterm birth to inhibit uterine contractions. Nifedipine is a calcium channel blocking agent used to delay threatened spontaneous preterm birth, however, has limited efficacy and lacks preclinical data regarding mechanisms of action. It is unknown if nifedipine affects the pro-inflammatory environment associated with preterm labour pathophysiology and we hypothesise nifedipine only targets myometrial contraction rather than also mitigating inflammation. We assessed anti-inflammatory and anti-contractile effects of nifedipine on human myometrium using in vitro and ex vivo techniques, and a mouse model of preterm birth. We show that nifedipine treatment inhibited contractions in myometrial in vitro contraction assays (P = 0.004 vs. vehicle control) and potently blocked spontaneous and oxytocin-induced contractions in ex vivo myometrial tissue in muscle myography studies (P = 0.01 vs. baseline). Nifedipine treatment did not reduce gene expression or protein secretion of pro-inflammatory cytokines in either cultured myometrial cells or ex vivo tissues. Although nifedipine could delay preterm birth in some mice, this was not consistent in all dams and was overall not statistically significant. Our data suggests nifedipine does not modulate preterm birth via inflammatory pathways in the myometrium, and this may account for its limited clinical efficacy. [ABSTRACT FROM AUTHOR]

Preeclampsia is associated with an increased lifelong risk of cardiovascular disease (CVD). It is not clear whether this is induced by persistent systemic organ and vascular damage following preeclampsia or due to a predisposition to both conditions that share cardiovascular pathophysiology. Common to both CVD and preeclampsia is the dysregulation of corin and its proteolytic product, atrial natriuretic peptide (ANP). ANP, a hypotensive hormone converted from pro-ANP by corin, is involved in blood pressure homeostasis. While corin is predominantly a cardiac enzyme, both corin and pro-ANP are significantly upregulated in the gravid uterus and dysregulated in preeclampsia. Relatively little is known about ANP function in the endothelium during a pregnancy complicated by preeclampsia. Here, we investigated the effect of ANP on endothelial cell proliferation and migration, markers of endothelial dysfunction, and receptor expression in omental arteries exposed to circulating preeclamptic toxins. ANP receptor expression is significantly upregulated in preeclamptic vasculature but not because of exposure to preeclampsia toxins tumour necrosis factor α or soluble fms-like tyrosine kinase-1. The supplementation of endothelial cells with ANP did not promote proliferation or migration, nor did ANP improve markers of endothelial dysfunction. The role of ANP in preeclampsia is unlikely to be via endothelial pathways. [ABSTRACT FROM AUTHOR]

Background:We have recently shown first trimester Macrophage inhibitory cytokine-1 (MIC-1) and Pregnancy Associated Plasma Protein-A (PAPP-A) serum concentrations are depressed among asymptomatic women destined to miscarry. Here we examined whether plasma levels of MIC-1 and PAPP-A are depressed among women presenting to an Early Pregnancy Assessment Unit (EPAU), noted to have a confirmed viable fetus, but subsequently miscarry. Methods:We performed a prospective cohort study, recruiting 462 women in the first trimester presenting to EPAU and had fetal viability confirmed by ultrasound. We obtained plasma samples on the same day and measured MIC-1, PAPP-A and human chorionic gonadotrophin (hCG), grouping the cohort according to whether they later miscarried or not. To correct for changes in analyte levels across gestation, we expressed the data as Multiples of the normal Median (MoMs). Results:We recruited 462 participants presenting to EPAU at 5-12 weeks gestation. Most (80%) presented with symptoms of threatened miscarriage (e.g. abdominal pain, vaginal bleeding). 34 (7.4%) subsequently miscarried. Median plasma MIC-1 levels among those who miscarried were 50% of those with ongoing pregnancies (Miscarriage cohort MoM 0.50 (25th-75th centiles: 0.29-1.33) vs ongoing pregnancies MoM 1.00 (0.65-1.38); p=0.0025). Median plasma PAPP-A MoMs among those who miscarried was 0.57 (0.00-1.12), significantly lower than those with ongoing pregnancies (MoMs 1.00 (0.59-1.59); p=0.036). Plasma hCG levels were also significantly depressed among those who miscarried compared to those with ongoing pregnancies. However, the performance of MIC-1 as a diagnostic marker to predict miscarriage in this cohort was modest, and not improved with the addition of hCG. Conclusion:MIC-1 and PAPP-A levels are significantly depressed in women presenting to EPAU with ultrasound evidence of fetal viability, but later miscarry. While they are unlikely to be useful as predictive biomarkers in this clinical setting, they probably play important roles in the maintenance of early pregnancy. [ABSTRACT FROM AUTHOR]

Placental dysfunction is the leading cause of both preeclampsia and fetal growth restriction. This study aimed to characterize endothelial protein C receptor (EPCR) in preterm preeclampsia, term preeclampsia, and fetal growth restriction (defined by delivery of a small for gestational age [SGA] infant [<10% birthweight centile]) and examine its regulation in primary syncytiotrophoblast. Placental EPCR mRNA and protein were significantly increased in patients with preterm preeclampsia (<34 weeks gestation) compared to gestation‐matched controls (p <.0001). In the plasma, EPCR was also significantly elevated (p =.01) in established preterm preeclampsia while its substrate, protein C (PC) was significantly reduced (p =.0083). Placentas from preterm small for gestational age (SGA) cases, had elevated EPCR mRNA expression (p <.0001) relative to controls. At 36 weeks, no significant changes in plasma EPCR were detected in samples from patients destined to develop preeclampsia or deliver an SGA infant at term. In terms of syncytiotrophoblast, hypoxia significantly increased EPCR mRNA expression (p =.008), but Tumor Necrosis Factor Alpha (TNF‐α) decreased EPCR mRNA. Interleukin‐6 (IL‐6) had no significant effect on EPCR mRNA expression. When isolated syncytiotrophoblast was treated with metformin under hypoxia (1% O2) or normoxia (8% O2), EPCR mRNA expression was significantly reduced (p =.008) relative to control. In conclusion, EPCR is markedly elevated in the placenta and the circulation of patients with established preterm preeclampsia and placental increases may be associated with hypoxia. Additionally, fetal growth‐restricted pregnancies (as defined by the delivery of an SGA infant) also demonstrated elevated placental EPCR. [ABSTRACT FROM AUTHOR]

Background: Cell-free RNA (cfRNA) transcripts known to be expressed by the fetal brain are detectable by quantitative reverse transcription PCR (RT-qPCR) in amniotic fluid and represent potential biomarkers of neurodevelopment. The aim of this study was to compare the cfRNA yields from amniotic fluid (AF) collected in a commercial RNA stabilization product with the traditional method of freezing alone. Findings: Thirteen women undergoing elective Cesarean birth at term without labor had whole AF collected at the time of uterine incision, prior to membrane rupture. Patient samples were split between Streck RNA blood collection tubes (BCT) and plain sterile polypropylene centrifuge tubes. Cell-free RNA from the AF supernatant was extracted according to a previously published protocol. RT qPCR was performed for the reference gene GAPDH, and three genes associated with neurodevelopment (NRXN3, NTRK3, and ZBTB18). The yield from samples collected in Streck RNA BCT and plain centrifuge tubes were compared with the paired t test. GAPDH, NRXN3 and ZBTB18 amplified successfully in all samples, but NTRK3 did not. The RNA yield was significantly lower in samples collected in the Streck RNA BCT compared with the traditional storage method of freezing alone for all three successfully amplified genes (p < 0.0001). Conclusions: Selected cfRNA neurodevelopment transcripts are consistently detectable in third trimester AF. There appears to be no benefit in collecting AF in Streck RNA BCT for quantitative studies of AF cell-free RNA. [ABSTRACT FROM AUTHOR]

New-onset maternal hypertension is a hallmark of preeclampsia, driven by widespread endothelial dysfunction and systemic vasoconstriction. Here, we set out to create a new ex vivo model using preeclamptic serum to cause injury to the endothelium, mimicking vascular dysfunction in preeclampsia and offering the potential to evaluate candidate therapeutic interventions. Human omental arteries were collected at caesarean section from normotensive pregnant patients at term (n = 9). Serum was collected from pregnancies complicated by preterm preeclampsia (birth < 34 weeks' gestation, n = 16), term preeclampsia (birth > 37 weeks' gestation, n = 5), and healthy gestation-matched controls (preterm n = 16, term n = 12). Using wire myography, we performed ex vivo whole vessel assessment where human omental arteries were treated with increasing doses of each serum treatment (2–20%) and vasoreactivity was assessed. All pregnant serum treatments successfully drove vasoconstriction; no significant difference was observed in the degree of vasoconstriction when exposed to preeclamptic or control serum. We further demonstrated the ability of esomeprazole (a candidate therapeutic for preeclampsia; 0.1–100 µM) to drive vasorelaxation of pre-constricted vessels (only with serum from preeclamptic patients). In summary, we describe a novel human physiological model of preeclamptic vascular constriction. We demonstrate its exciting potential to screen drugs for their therapeutic potential as treatment for vasoconstriction induced by preeclampsia. [ABSTRACT FROM AUTHOR]

Previously, we demonstrated that the proton pump inhibitor, esomeprazole magnesium hydrate (MH), could have potential as a repurposed treatment against preeclampsia, a serious obstetric condition. In this study we investigate the difference in the preclinical effectiveness between 100 µM of esomeprazole MH and its hydration isomer, esomeprazole magnesium trihydrate (MTH). Here, we found that both treatments reduced secretion of sFLT-1 (anti-angiogenic factor) from primary cytotrophoblast, but only esomeprazole MH reduced sFLT-1 secretion from primary human umbilical vein endothelial cells (assessed via ELISA). Both drugs could mitigate expression of the endothelial dysfunction markers, vascular cell adhesion molecule-1 and endothelin-1 (via qPCR). Neither esomeprazole MH nor MTH quenched cytotrophoblast reactive oxygen species production in response to sodium azide (ROS assay). Finally, using wire myography, we demonstrated that both compounds were able to induce vasodilation of human omental arteries at 100 µM. Esomeprazole is safe to use in pregnancy and a candidate treatment for preeclampsia. Using primary human tissues and cells, we validated that esomeprazole is effective in enhancing vascular relaxation, and can reduce key factors associated with preeclampsia, including sFLT-1 and endothelial dysfunction. However, esomeprazole MH was more efficacious than esomeprazole MTH in our in vitro studies. [ABSTRACT FROM AUTHOR]

Preeclampsia is a devastating, multisystem disorder of pregnancy. It has no cure except delivery, which if premature can impart significant neonatal morbidity. Efforts to repurpose pregnancy-safe therapeutics for the treatment of preeclampsia have led to the assessment of the proton pump inhibitor, esomeprazole. Preclinically, esomeprazole reduced placental secretion of anti-angiogenic sFlt-1, improved endothelial dysfunction, promoted vasorelaxation, and reduced maternal hypertension in a mouse model. Our understanding of the precise mechanisms through which esomeprazole works to reduce endothelial dysfunction and enhance vasoreactivity is limited. Evidence from earlier studies suggested esomeprazole might work via the nitric oxide pathway, upregulating endothelial nitric oxide synthase (eNOS). Here, we investigated the effect of esomeprazole in a mouse model of L-NAME-induced hypertension (decreased eNOS activity). We further antagonised the model by addition of diet-induced obesity, which is relevant to both preeclampsia and the nitric oxide pathway. Esomeprazole did not decrease blood pressure in this model, nor were there any alterations in vasoreactivity or changes in foetal outcomes in lean mice. We observed similar findings in the obese mouse cohort, except esomeprazole treatment enhanced ex vivo acetylcholine-induced vasorelaxation. As acetylcholine induces nitric oxide production, these findings hint at a function for esomeprazole in the nitric oxide pathway. [ABSTRACT FROM AUTHOR]

Background: We have previously shown in two independent cohorts that circulating first trimester Macrophage Inhibitory Cytokine-1 (MIC-1) levels are lower in women in early pregnancy who are destined to miscarriage. While promising, the diagnostic performance of measuring MIC-1 alone was not sufficient for it to be a useful predictive test for miscarriage. Besides MIC-1, there are other cytokines, as well as chemokines, involved in facilitating early pregnancy. We reasoned that screening these factors in maternal plasma could uncover other predictive markers of miscarriage. Methods: This was a nested case control study, of 78 women from a prospective study of 462 attending the Early Pregnancy Assessment Unit in the first trimester (EPAU) with a threatened miscarriage; 34 of these subsequently miscarried (cases) and 44 went on to have a normal delivery (controls) Cytokines IL-1β, IL-6 and IL-10, and the chemokines, CXCL8, CCL2, CCL5, CCL7 and CX3CL1 were measured in plasma from our cohort. Results: The cytokines IL-1β, IL-6, IL-10 and the chemokine CXCL8 were not detectable in first trimester plasma. The chemokines CCL2, CCL5, CCL7 and CX3CL1 were detectable in all samples but levels did not vary across 5–12 weeks of gestation among controls. Plasma levels of these chemokines were no different in the miscarriage cohort compared to controls. Conclusion: The chemokines CCL2, CCL5, CCL7 and CX3CL1 were detectable in plasma during the first trimester while IL-1β, IL-6, IL-10 and CXCL8 were not. However, none of the cytokines and chemokines screened were different in maternal plasma in cases or controls. These therefore do not appear to have potential for application as predictive biomarkers of miscarriage. [ABSTRACT FROM AUTHOR]

Background: Fetal growth restriction is a disorder of placental dysfunction with three to four-fold increased risk of stillbirth. Fetal growth restriction has pathophysiological features in common with preeclampsia. We hypothesised that angiogenesis-related factors in maternal plasma, known to predict preeclampsia, may also detect fetal growth restriction at 36 weeks' gestation. We therefore set out to determine the diagnostic performance of soluble fms-like tyrosine kinase 1 (sFlt-1), placental growth factor (PlGF), and the sFlt-1:PlGF ratio, measured at 36 weeks' gestation, in identifying women who subsequently give birth to small-for-gestational-age (SGA; birthweight <10th centile) infants. We also aimed to validate the predictive performance of the analytes for late-onset preeclampsia in a large independent, prospective cohort.Methods: A nested 1:2 case-control study was performed including 102 cases of SGA infants and a matched group of 207 controls; and 39 cases of preeclampsia. We determined the diagnostic performance of each angiogenesis-related factor, and of their ratio, to detect SGA infants or preeclampsia, for a predetermined 10% false positive rate.Results: Median plasma levels of PlGF at 36 weeks' gestation were significantly lower in women who subsequently had SGA newborns (178.5 pg/ml) compared to normal birthweight controls (326.7 pg/ml, p < 0.0001). sFlt-1 was also higher among SGA cases, but this was not significant after women with concurrent preeclampsia were excluded. The sensitivity of PlGF to predict SGA infants was 28.8% for a 10% false positive rate. The sFlt-1:PlGF ratio demonstrated better sensitivity for preeclampsia than either analyte alone, detecting 69.2% of cases for a 10% false positive rate.Conclusions: Plasma PlGF at 36 weeks' gestation is significantly lower in women who subsequently deliver a SGA infant. While the sensitivity and specificity of PlGF currently limit clinical translation, our findings support a blood-based biomarker approach to detect late-onset fetal growth restriction. Thirty-six week sFlt-1:PlGF ratio predicts 69.2% of preeclampsia cases, and could be a useful screening test to triage antenatal surveillance. [ABSTRACT FROM AUTHOR]

Nuclear Receptor Subfamily 4 Group A Member 2 (NR4A2) transcripts are elevated in the circulation of individuals whose pregnancies are complicated by preterm fetal growth restriction (FGR). In this paper, we show that the cases with preeclampsia (PE) have increased circulating NR4A2 transcripts compared to those with normotensive FGR. We aimed to establish whether the dysfunctional placenta mirrors the increase in NR4A2 transcripts and further, to uncover the function of placental NR4A2. NR4A2 expression was detected in preterm and term placental tissue; expressed higher at term. NR4A2 mRNA expression and protein were not altered in placentas from preterm FGR or PE pregnancies. Hypoxia (1% O2 compared to 8% O2) significantly reduced cytotrophoblast NR4A2 mRNA expression, but not placental explant NR4A2 expression. Silencing cytotrophoblast NR4A2 expression under hypoxia (via short interfering (si)RNAs) did not alter angiogenic Placental Growth Factor, nor anti-angiogenic sFlt-1 mRNA expression or protein secretion, but increased expression of cellular antioxidant, oxidative stress, inflammatory, and growth genes. NR4A2 expression was also not altered in a model of tumour necrosis factor-α-induced endothelial dysfunction, or with pravastatin treatment. Further studies are required to identify the origin of the circulating transcripts in pathological pregnancies, and investigate the function of placental NR4A2. [ABSTRACT FROM AUTHOR]

Introduction: Poor gestational outcomes due to placental insufficiency can have lifelong consequences for mother and child. Objective: There is a need for better methods of diagnosis, and elemental metabolomics may provide a means to determine the risk of gestational disorders. Methods: This study used blood plasma samples collected at 36 weeks' gestation from women who later developed preeclampsia (n = 38), or small-for-gestational age babies (n = 91), along with matched controls (n = 193). Multi-element analysis was conducted by inductively coupled plasma mass spectrometer (ICP-MS), allowing simultaneous measurement of 28 elements. Results: Women who later developed PE, exhibited significantly increased concentrations of K, Rb and Ba. For SGA pregnancies, there was a significant increase in Cu and a decrease in As concentrations. Despite significant differences in single elements, the elemental profile of groups indicated no clustering of control, PE, or SGA samples. Positive predicative values correctly identified approximately 60% of SGA and 70% of PE samples. Conclusion: This is the first-time elemental metabolomics has been used to predict SGA and PE at 36 weeks. Though significant changes were identified, routine clinical use may be limited but may contribute to a multi marker test. Future analysis should include other biomarkers, metabolic data or clinical measurements made throughout gestation. [ABSTRACT FROM AUTHOR]

Previously, we identified increased maternal circulating DAAM2 mRNA in pregnancies complicated by preterm fetal growth restriction (FGR). Here, we assessed whether circulating DAAM2 mRNA could detect FGR, and whether the DAAM2 gene, known to play roles in the Wnt signalling pathway is expressed in human placenta and associated with dysfunction and FGR. We performed linear regression analysis to calculate area under the ROC curve (AUC) for DAAM2 mRNA expression in the maternal circulation of pregnancies complicated by preterm FGR. DAAM2 mRNA expression was assessed across gestation by qPCR. DAAM2 protein and mRNA expression was assessed in preterm FGR placenta using western blot and qPCR. DAAM2 expression was assessed in term cytotrophoblasts and placental explant tissue cultured under hypoxic and normoxic conditions by qPCR. Small interfering RNAs were used to silence DAAM2 in term primary cytotrophoblasts. Expression of growth, apoptosis and oxidative stress genes were assessed by qPCR. Circulating DAAM2 mRNA was elevated in pregnancies complicated by preterm FGR [p < 0.0001, AUC = 0.83 (0.78–0.89)]. Placental DAAM2 mRNA was detectable across gestation, with highest expression at term. DAAM2 protein was increased in preterm FGR placentas but demonstrated no change in mRNA expression. DAAM2 mRNA expression was increased in cytotrophoblasts and placental explants under hypoxia. Silencing DAAM2 under hypoxia decreased expression of pro-survival gene, BCL2 and oxidative stress marker, NOX4, whilst increasing expression of antioxidant enzyme, HMOX-1. The increased DAAM2 associated with FGR and hypoxia implicates a potential role in placental dysfunction. Decreasing DAAM2 may have cytoprotective effects, but further research is required to elucidate its role in healthy and dysfunctional placentas. [ABSTRACT FROM AUTHOR]

Background: Fetuses affected by placental insufficiency do not receive adequate nutrients and oxygenation, become growth restricted and acidemic, and can demise. Preterm fetal growth restriction is a severe form of placental insufficiency with a high risk of stillbirth. We set out to identify maternal circulating mRNA transcripts that are differentially expressed in preterm pregnancies complicated by very severe placental insufficiency, in utero fetal acidemia, and are at very high risk of stillbirth.Methods: We performed a cohort study across six hospitals in Australia and New Zealand, prospectively collecting blood from 128 pregnancies complicated by preterm fetal growth restriction (delivery < 34 weeks' gestation) and 42 controls. RNA-sequencing was done on all samples to discover circulating mRNAs associated with preterm fetal growth restriction and fetal acidemia in utero. We used RT-PCR to validate the associations between five lead candidate biomarkers of placental insufficiency in an independent cohort from Europe (46 with preterm fetal growth restriction) and in a third cohort of pregnancies ending in stillbirth.Results: In the Australia and New Zealand cohort, we identified five mRNAs that were highly differentially expressed among pregnancies with preterm fetal growth restriction: NR4A2, EMP1, PGM5, SKIL, and UGT2B1. Combining three yielded an area under the receiver operative curve (AUC) of 0.95. Circulating NR4A2 and RCBTB2 in the maternal blood were dysregulated in the presence of fetal acidemia in utero. We validated the association between preterm fetal growth restriction and circulating EMP1, NR4A2, and PGM5 mRNA in a cohort from Europe. Combining EMP1 and PGM5 identified fetal growth restriction with an AUC of 0.92. Several of these genes were differentially expressed in the presence of ultrasound parameters that reflect placental insufficiency. Circulating NR4A2, EMP1, and RCBTB2 mRNA were differentially regulated in another cohort destined for stillbirth, compared to ongoing pregnancies. EMP1 mRNA appeared to have the most consistent association with placental insufficiency in all cohorts.Conclusions: Measuring circulating mRNA offers potential as a test to identify pregnancies with severe placental insufficiency and at very high risk of stillbirth. Circulating mRNA EMP1 may be promising as a biomarker of severe placental insufficiency. [ABSTRACT FROM AUTHOR]

Pre-eclampsia is a leading cause of maternal and fetal morbidity and mortality. Characterised by the onset of hypertension and proteinuria in the second half of pregnancy, it can lead to maternal end-organ injury such as cerebral ischemia and oedema, pulmonary oedema and renal failure, and potentially fatal outcomes for both mother and fetus. The causes of the different maternal end-organ phenotypes of pre-eclampsia and why some women develop pre-eclampsia condition early in pregnancy have yet to be elucidated. Omics methods include proteomics, genomics, metabolomics, transcriptomics. These omics techniques, previously mostly used on bulk tissue and individually, are increasingly available at a single cellular level and can be combined with each other. Multi-omics techniques on a single-cell or spatial level provide us with a powerful tool to understand the pathophysiology of pre-eclampsia. This review will explore the status of omics methods and how they can and could contribute to understanding the pathophysiology of pre-eclampsia. • The pathophysiology of pre-eclampsia and ist subtypes remains to be elucidated. • Various omics techniques are increasingly available on a single-cell and spatial level. • These novel techniques may help to better understand the pathophysiology of pre-eclampsia and its different phenotypes. [ABSTRACT FROM AUTHOR]

Preeclampsia is one of the most serious complications of pregnancy. Currently there are no medical treatments. Given placental oxidative stress may be an early trigger in the pathogenesis of preeclampsia, therapies that enhance antioxidant pathways have been proposed as treatments. Melatonin is a direct free-radical scavenger and indirect antioxidant. We performed in vitro assays to assess whether melatonin 1) enhances the antioxidant response element genes (heme-oxygenase 1, (HO-1), glutamate-cysteine ligase (GCLC), NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1), thioredoxin (TXN)) or 2) alters secretion of the anti-angiogenic factors soluble fms-like tyrosine kinase-1 (sFLT) or soluble endoglin (sENG) from human primary trophoblasts, placental explants and human umbilical vein endothelial cells (HUVECs) and 3) can rescue TNF-α induced endothelial dysfunction. In primary trophoblast melatonin treatment increased expression of the antioxidant enzyme TXN. Expression of TXN, GCLC and NQO1 was upregulated in placental tissue with melatonin treatment. HUVECs treated with melatonin showed an increase in both TXN and GCLC. Melatonin did not increase HO-1 expression in any of the tissues examined. Melatonin reduced sFLT secretion from primary trophoblasts, but had no effect on sFLT or sENG secretion from placental explants or HUVECs. Melatonin did not rescue TNF-α induced VCAM-1 and ET-1 expression in endothelial cells. Our findings suggest that melatonin induces antioxidant pathways in placenta and endothelial cells. Furthermore, it may have effects in reducing sFLT secretion from trophoblast, but does not reduce endothelial dysfunction. Given it is likely to be safe in pregnancy, it may have potential as a therapeutic agent to treat or prevent preeclampsia. [ABSTRACT FROM AUTHOR]

Preeclampsia is a hypertensive disorder of pregnancy, responsible for over 60 000 maternal deaths annually. Endothelial dysfunction is a central aspect to its pathophysiology, and currently, no medical therapeutic is available for its treatment. In this study, we aim to investigate the effect of epidermal growth factor (EGF) on endothelial dysfunction using primary human tissues. We performed a number of in vitro assays that mimic the vascular endothelial dysfunction that occurs in preeclampsia. Epidermal growth factor reduced the expression of vascular cell adhesion molecule-1, a marker of endothelial dysfunction, after insult with tumor necrosis factor α (TNF-α) or serum from women with preeclampsia. Additionally, after TNF-α insult, EGF reduced tube disruption and the adhesion of monocytes to primary human umbilical vein endothelial cells (HUVECs). Our findings suggest that EGF reduces endothelial dysfunction in primary HUVECs. Epidermal growth factor may have potential as a novel peptide treatment for preeclampsia and other diseases where there is endothelial dysfunction. [ABSTRACT FROM AUTHOR]

Embryo implantation requires synchronized dialogue between the receptive endometrium and activated blastocyst via locally produced soluble mediators. During the mid-secretory (MS) phase of the menstrual cycle, increased glandular secretion into the uterine lumen provides important mediators that modulate the endometrium and support the conceptus during implantation. Previously we demonstrated the importance of vascular endothelial growth factor (VEGF) in the human uterus, particularly with respect to embryo implantation. In the current study, proteomic analysis of human uterine lavage fluid identified the presence of placental growth factor (PlGF) a homolog of VEGF, that binds the VEGF receptor 1 (VEGFR1). Analysis of immunostaining for PlGF in human endometrial tissue across the menstrual cycle (from both fertile and infertile women) revealed PlGF was predominantly localised to glandular and luminal epithelial cells, with staining in the decidualising stromal cells surrounding the maternal spiral arteries in the secretory phase of the menstrual cycle. Immunoreactive PlGF was also detected in subpopulations of endometrial leukocytes. Functional studies demonstrated that culturing mouse embryos with recombinant human (rh)PlGF enhanced blastocyst cell number and outgrowth. Furthermore, treatment of human endometrial epithelial cells (EEC) with rhPlGF enhanced EEC adhesion. Taken together, these data demonstrate that PlGF is abundant in the human endometrium, and secreted into the uterine lumen where it mediates functional changes in cellular adhesion with important roles in implantation. [ABSTRACT FROM AUTHOR]

Background: Preeclampsia is associated with the placental release of soluble fms-like tyrosine kinase 1 (sFlt-1) and soluble endoglin (sENG). These anti-angiogenic factors cause hypertension and multi-organ injury. Pravastatin decreases placental secretion of sFlt-1 in vitro and is currently being examined in clinical trials as a potential treatment for preeclampsia. However, it is possible that different classes of statins may be more potent at decreasing sFlt-1 secretion. We compared the relative potency of three different generations of statins on sFlt-1 and sENG secretion from human endothelial cells, trophoblast cells, and placenta explants.Methods: We performed functional experiments using primary human umbilical vein endothelial cells, trophoblast cells and preterm preeclamptic placental explants to assess the affect of simvastatin, rosuvastatin and pravastatin on sFlt-1 and sENG secretion and compared the relative potency of each statin at reducing these factors (Inhibitory Concentration 50). Furthermore we assessed the effect of each statin on the antioxidant and cytoprotective enzyme, heme-oxygenase 1.Results: All statins reduced sFlt-1 secretion from endothelial cells, trophoblasts and preterm preeclamptic placental explants. Simvastatin was the most potent inhibitor of sFlt-1 secretion from endothelial cells (IC 50 3.2 μM), trophoblast cells (IC 50 61.4 μM) and placental explants. Simvastatin was 28 times and 3 times more potent at reducing sFlt-1 secretion from endothelial cells and 85 times and 33 times more potent at reducing sFlt-1 secretion from trophoblast cells than pravastatin or rosuvastatin respectively. All statins increased sENG secretion from endothelial cells however did not change secretion from placental explants. While all statins up-regulated heme-oxygenase 1 in endothelial cells, only simvastatin up-regulated its expression in placenta from patients with preterm preeclampsia.Conclusion: Simvastatin may be a more potent inhibitor of sFlt-1 secretion from endothelial cells, trophoblast cells and placenta from women with preterm preeclampsia than either pravastatin or rosuvastatin. [ABSTRACT FROM AUTHOR]

Preeclampsia (PE) and intrauterine growth restriction (IUGR) are major obstetric health problems. Higher levels of T-helper (Th) 1 (proinflammatory) cytokines have been observed in pregnancies complicated with PE and IUGR; this is in contrast to the predominant Th2 (anti-inflammatory) cytokine environment found in uncomplicated pregnancies. Myostatin is best known as a negative regulator of muscle development and reportedly has a role in fat deposition, glucose metabolism, and cytokine modulation (outside the placenta). Myostatin concentrations in plasma and protein expression in placental tissue are significantly higher in women with PE. Expression of myostatin in IUGR and PE-IUGR and the effect of this protein on the cytokine production from the placenta is unknown. In the current study, significant differences were identified in the expression of myostatin in pregnancies complicated with IUGR, PE, and PE with IUGR. Furthermore, cytokine production by first-trimester placental tissues was altered following myostatin treatment. [ABSTRACT FROM AUTHOR]

Preeclampsia is a serious complication of pregnancy with no medical treatment. It is caused by intermittent placental hypoxia and release of sFlt-1 and soluble endoglin, leading to wide spread maternal endothelial dysfunction and multisystem organ injury. YC-1 is a guanylyl cyclase activator and HIF1 α inhibitor developed for use in hypertension and atherosclerosis. We examined whether YC-1 reduces sFlt-1 and sENG secretion and reverses endothelial dysfunction in primary human tissues. YC-1 significantly reduced sFlt-1 and sENG secretion from human umbilical vein endothelial cells, purified primary trophoblast cells and placental explants taken from patients with preterm preeclampsia. This was concordant with reduced HIF1 α expression. YC-1 also reversed TNFα induced endothelial dysfunction, including reduced vascular cell adhesion molecule 1 expression and monocyte adhesion to primary endothelial cells. We conclude YC-1 decreases placental production of sFlt-1 and sENG and decreases endothelial dysfunction. It is a novel therapeutic candidate for preeclampsia. [ABSTRACT FROM AUTHOR]

Background: Preterm prelabor rupture of the fetal membranes (PPROM) is a significant contributor to the morbidity and mortality of preterm birth, particularly in the setting of chorioamnionitis. No sensitive or specific diagnostic or predictive test currently exists for the accurate diagnosis of chorioamnionitis. Our aim was to measure messenger RNA (mRNA) coding cytokines in the maternal blood and examine whether they were increased in association with chorioamnionitis at delivery. Methods/Results: We performed a prospective cohort study of women recruited with PPROM at a mean gestational age of 28.9 weeks at risk of developing chorioamnionitis. Blood was sampled from participants, and the expression of mRNA coding for proinflammatory genes was measured in women with and without chorioamnionitis at the time of delivery as well as gestation-matched healthy controls. Expression was measured using quantitative polymerase chain reaction (PCR) and also digital PCR. Interleukin 1β (IL1B) mRNA expression in maternal blood was elevated in women with chorioamnionitis compared to gestation-matched controls. Importantly, among women admitted with PPROM, digital PCR confirmed a significant increase in IL1B expression in maternal blood in women with chorioamnionitis compared to women without chorioamnionitis. Polymerase chain reaction array revealed that CD14, nuclear factor of κ light polypeptide gene enhancer in B-cells 1 (NFKB1), and tumor necrosis factor receptor super family-interacting serine-threonine kinase 1 mRNA were significantly increased in women with chorioamnionitis compared to controls. Digital PCR confirmed that NFKB1 mRNA was significantly increased in patients with chorioamnionitis compared to controls and that CD14 levels increased over time in patients with PPROM having chorioamnionitis. Conclusion: Measuring circulating proinflammatory mRNA in women with PPROM may distinguish those with chorioamnionitis from those without, in turn providing better targeted therapies and appropriate timing of delivery. [ABSTRACT FROM AUTHOR]

Severe early onset pre-eclampsia is a serious pregnancy complication, believed to arise as a result of persistent placental hypoxia due to impaired placentation. Pregnancy-associated plasma protein A2 (PAPPA2) is very highly expressed in the placenta relative to all other tissues. There is some evidence that PAPPA2 mRNA and protein are increased in association with pre-eclampsia. The aim of the present study was to characterise the mRNA and protein expression, as well as localisation, of PAPPA2 in an independent cohort of severe early onset pre-eclamptic placentas. We also examined whether exposing placental explants to hypoxia (1% oxygen) changed the expression of PAPPA2. Expression of PAPPA2 mRNA and protein was upregulated in severe early onset pre-eclamptic placentas compared with preterm controls and localised to the syncytiotrophoblast. Interestingly, protein localisation was markedly reduced in term placenta. Syncytialisation of BeWo cells did not change PAPPA2 expression. However, hypoxia upregulated PAPPA2 mRNA and protein expression in primary placental explants. Together, our data suggest that PAPPA2 may be upregulated in severe pre-eclampsia and, functionally, this may be mediated via increased placental hypoxia known to occur with this pregnancy disorder. [ABSTRACT FROM AUTHOR]

Fetal growth restriction (FGR), when undetected antenatally, is the biggest risk factor for preventable stillbirth. Maternal circulating SPINT1 is reduced in pregnancies, which ultimately deliver small for gestational age (SGA) infants at term (birthweight < 10th centile), compared to appropriate for gestational age (AGA) infants (birthweight ≥ 10th centile). SPINT1 is also reduced in FGR diagnosed before 34 weeks' gestation. We hypothesised that circulating SPINT1 would be decreased in co-existing preterm preeclampsia and FGR. Plasma SPINT1 was measured in samples obtained from two double-blind, randomised therapeutic trials. In the Preeclampsia Intervention with Esomeprazole trial, circulating SPINT1 was decreased in women with preeclampsia who delivered SGA infants (n = 75, median = 18,857 pg/mL, IQR 10,782–29,890 pg/mL, p < 0.0001), relative to those delivering AGA (n = 22, median = 40,168 pg/mL, IQR 22,342–75,172 pg/mL). This was confirmed in the Preeclampsia Intervention 2 with metformin trial where levels of SPINT1 in maternal circulation were reduced in SGA pregnancies (n = 95, median = 57,764 pg/mL, IQR 42,212–91,356 pg/mL, p < 0.0001) compared to AGA controls (n = 40, median = 107,062 pg/mL, IQR 70,183–176,532 pg/mL). Placental Growth Factor (PlGF) and sFlt-1 were also measured. PlGF was significantly reduced in the SGA pregnancies, while ratios of sFlt-1/SPINT1 and sFlt1/PlGF were significantly increased. This is the first study to demonstrate significantly reduced SPINT1 in co-existing FGR and preeclamptic pregnancies. [ABSTRACT FROM AUTHOR]

Background: Ectopic pregnancy (EP) occurs in 1–2% of pregnancies, but is over-represented as a leading cause of maternal death in early pregnancy. It remains a challenge to diagnose early and accurately. Women often present in early pregnancy with a ‘pregnancy of unknown location’ (PUL) and the diagnosis and exclusion of EP is difficult due to a lack of reliable biomarkers. A serum biomarker able to clearly distinguish between EP and other pregnancy outcomes would greatly assist clinicians in diagnosing and safely managing PULs. This study evaluates the ability of maternal serum macrophage inhibitory cytokine-1 (MIC-1) levels to differentiate between EP and other pregnancy outcomes in women with a PUL. Methods: Sera were collected from 120 women with a PUL at first clinical presentation and assayed for MIC-1 by ELISA. Results were classified according to ultimate pregnancy outcome and the discriminatory ability of MIC-1 to diagnose EP was assessed. Results: Serum MIC-1 levels were lower in women with histologically confirmed (definite) EP (dEP) (median 552 ng/mL; interquartile range (IQR) 414–693 ng/mL) compared to women with definite viable intra-uterine pregnancies (dVIUPs) (722 ng/mL; IQR 412–1122 ng/mL), and higher when compared to women with definite non-viable intra-uterine pregnancies (dNVIUPs) (465 ng/mL; IQR 341–675 ng/mL). MIC-1 levels were significantly higher in women with dEP compared to women whose PULs resolved without medical intervention (srPUL) (401 ng/mL; IQR 315–475 ng/mL) (p<0.003). There were no women with an ectopic pregnancy where serum MIC-1>1000 ng/mL. Conclusion: Serum MIC-1 levels in PUL were not able to categorically diagnose EP, however, MIC-1 could distinguish women with an EP that required medical intervention and those women whose PULs spontaneously resolved. A single serum MIC-1 measurement also excluded EP at levels above 1000 ng/mL. MIC-1 may play a role in the development of a combined assay of biomarkers for the diagnosis of EP. [ABSTRACT FROM AUTHOR]

Background: There has been recent evidence suggesting the presence of anti-thyroid peroxidase antibodies (TPOAb) increases the risk of miscarriage, and levothyroxine can rescue miscarriages associated with TPOAb. We propose the most clinically pragmatic cohort to screen for TPOAb are women presenting for management of a missed miscarriage and have never birthed a liveborn. We measured serum TPOAb among nulliparous women presenting for management of miscarriage, and compared levels with women who have had 2 or more livebirths (and never miscarried). Given its potential role in immunomodulation, we also measured Vitamin D levels. Methods: We performed a prospective descriptive cohort study at a tertiary hospital (Mercy Hospital for Women, Victoria, Australia). We measured TPOAb and Vitamin D levels in serum obtained from 118 nulliparous women presenting for management of miscarriage, and 162 controls with 2 or more livebirths (and no miscarriages). Controls were selected from a serum biobank prospectively collected in the first trimester at the same hospital. Results: Nulliparous women with 1 or more miscarriages had higher thyroid peroxidase antibody (TPOAb) levels than those with 2 or more livebirths; TPOAb in miscarriage group was 0.3 mIU/L (interquartile range [IR]: 0.2-0.7) vs 0.2 mIU/L among controls (IR 0.0-0.5; p < 0.0001). We confirmed TPOAb levels were not correlated with serum human chorionic gonadotrophin (hCG) concentrations in either the miscarriage or control groups. In contrast, thyroid stimulating hormone, fT3 and fT4 levels (thyroid hormones) either trended towards a correlation, or were significantly correlated with serum hCG levels in the two groups. Of the entire cohort that was predominantly caucasian, only 12% were Vitamin D sufficient. Low Vitamin D levels were not associated with miscarriage. Conclusions: We have confirmed the association between miscarriage and increased TPOAb levels. Furthermore, it appears TPOAb levels in maternal blood are not influenced by serum hCG levels. Therefore, we propose the day nulliparous women present for management for miscarriage is a clinically relevant, and pragmatic time to screen for TPOAb. [ABSTRACT FROM AUTHOR]

Background: Ectopic pregnancy (EP) remains the most life-threatening acute condition in modern gynaecology. It remains difficult to diagnose early and accurately. Women often present at emergency departments in early pregnancy with a 'pregnancy of unknown location' (PUL) and diagnosis/exclusion of EP is challenging due to a lack of reliable biomarkers. Recent studies suggest that serum levels of a disintegrin and metalloprotease protein-12 (ADAM-12) can be used differentiate EP from viable intrauterine pregnancy (VIUP). Here we describe a prospective study evaluating the performance of ADAM-12 in differentiating EP from the full spectrum of alternative PUL outcomes in an independent patient cohort. Methodology/Principal Findings: Sera were collected from 120 patients at their first clinical presentation with a PUL and assayed for ADAM-12 by ELISA. Patients were categorized according to final pregnancy outcomes. Serum ADAM-12 concentrations were increased in women with histologically-confirmed EP (median 442 pg/mL; 25%-75% percentile 232-783 pg/mL) compared to women with VIUP (256 pg/mL; 168-442 pg/mL) or miscarriage (192 pg/mL; 133-476 pg/mL). Serum ADAM-12 did not differentiate histologically- confirmed EP from spontaneously resolving PUL (srPUL) (416 pg/mL; 154-608 pg/mL). The diagnostic potential of ADAM-12 was only significant when 'ambiguous' PUL outcomes were excluded from the analysis (AROC = 0.6633; P = 0.03901). Conclusions/Significance: When measured in isolation, ADAM-12 levels had limited value as a diagnostic biomarker for EP in our patient cohort. The development of a reliable serum biomarker-based test for EP remains an ongoing challenge. [ABSTRACT FROM AUTHOR]

Despite significant maternal and fetal morbidity, a treatment for preeclampsia currently remains an unmet need in clinical care. As too does the lifelong cardiovascular risks imparted on preeclampsia sufferers. Endothelial dysfunction and end-organ injury are synonymous with both preeclampsia and cardiovascular disease, including heart failure. We propose that beta-blockers, known to improve endothelial dysfunction in the treatment of cardiovascular disease, and specifically known to reduce mortality in the treatment of heart failure, may be beneficial in the treatment of preeclampsia. Here, we assessed whether the beta-blockers carvedilol, bisoprolol, and metoprolol could quench the release of anti-angiogenic factors, promote production of pro-angiogenic factors, reduce markers of inflammation, and reduce endothelial dysfunction using our in vitro pre-clinical preeclampsia models encompassing primary placental tissue and endothelial cells. Here, we show beta-blockers effected a modest reduction in secretion of anti-angiogenic soluble fms-like tyrosine kinase-1 and soluble endoglin and increased expression of pro-angiogenic placental growth factor, vascular endothelial growth factor and adrenomedullin in endothelial cells. Beta-blocker treatment mitigated inflammatory changes occurring after endothelial dysfunction and promoted cytoprotective antioxidant heme oxygenase-1. The positive effects of the beta-blockers were predominantly seen in endothelial cells, with a less consistent response seen in placental cells/tissue. In conclusion, beta-blockers show potential as a novel therapeutic approach in the treatment of preeclampsia and warrant further investigation. [ABSTRACT FROM AUTHOR]

Biomarkers for placental dysfunction are currently lacking. We recently identified SPINT1 as a novel biomarker; SPINT2 is a functionally related placental protease inhibitor. This study aimed to characterise SPINT2 expression in placental insufficiency. Circulating SPINT2 was assessed in three prospective cohorts, collected at the following: (1) term delivery (n = 227), (2) 36 weeks (n = 364), and (3) 24–34 weeks' (n = 294) gestation. SPINT2 was also measured in the plasma and placentas of women with established placental disease at preterm (<34 weeks) delivery. Using first-trimester human trophoblast stem cells, SPINT2 expression was assessed in hypoxia/normoxia (1% vs. 8% O2), and following inflammatory cytokine treatment (TNFα, IL-6). Placental SPINT2 mRNA was measured in a rat model of late-gestational foetal growth restriction. At 36 weeks, circulating SPINT2 was elevated in patients who later developed preeclampsia (p = 0.028; median = 2233 pg/mL vs. controls, median = 1644 pg/mL), or delivered a small-for-gestational-age infant (p = 0.002; median = 2109 pg/mL vs. controls, median = 1614 pg/mL). SPINT2 was elevated in the placentas of patients who required delivery for preterm preeclampsia (p = 0.025). Though inflammatory cytokines had no effect, hypoxia increased SPINT2 in cytotrophoblast stem cells, and its expression was elevated in the placental labyrinth of growth-restricted rats. These findings suggest elevated SPINT2 is associated with placental insufficiency. [ABSTRACT FROM AUTHOR]

Preeclampsia is a pregnancy complication associated with elevated placental secretion of anti-angiogenic factors, maternal endothelial dysfunction and organ injury. GATA2 is a transcription factor expressed in the endothelium which regulates vascular homeostasis by controlling transcription of genes and microRNAs, including endothelial miR126. We assessed GATA2 and miR126 in preeclampsia. Whole blood circulating GATA2 mRNA and miR126 expression were significantly decreased in women with established early-onset preeclampsia compared to gestation-matched controls (p = 0.002, p < 0.0001, respectively). Using case-control groups selected from a large prospective cohort, whole blood circulating GATA2 mRNA at both 28 and 36 weeks' gestation was significantly reduced prior to the clinical diagnosis of preeclampsia (p = 0.012, p = 0.015 respectively). There were no differences in GATA2 mRNA or protein expression in preeclamptic placentas compared to controls, suggesting the placenta is an unlikely source. Inducing endothelial dysfunction in vitro by administering either tumour necrosis factor-α or placenta-conditioned media to endothelial cells, significantly reduced GATA2 mRNA expression (p < 0.0001), suggesting the reduced levels of circulating GATA2 mRNA may be of endothelial origin. Circulating GATA2 mRNA is decreased in women with established preeclampsia and decreased up to 12 weeks preceding onset of disease. Circulating mRNAs of endothelial origin may be a novel source of biomarker discovery for preeclampsia. [ABSTRACT FROM AUTHOR]