PAPP-A as a screening tool for assessment of gestational age before medication abortion in an intended-use population.

Introduction: Prior studies indicate that PAPP-A could serve as a marker of gestational age (GA) with the potential to determine eligibility for medication abortion. The authors validated the relationship between PAPP-A and GA in an actual-use population. Materials & methods: The authors collected blood samples, medical histories and ultrasound-determined GA from patients presenting for abortion services. They measured PAPP-A using two immunoassays and assessed diagnostic accuracy for predicting GA ≥71 days. Results: The Ansh Labs and R&D Systems immunoassays produced an area under the ROC curve of 0.982 (95% CI: 0.958-0.994) and 0.986 (95% CI: 0.963-0.996), respectively, for predicting GA ≥71 days. Conclusion: This validation study in an intended-use population confirmed that PAPP-A has a strong ability to distinguish pregnancies above and below 71 days' gestation. Clinical trial registration: NCT04232189 (ClinicalTrials.gov).

In the USA, abortion with pills is an option for people with pregnancies of less than 71 days. Usually, people use ultrasound to find out how far along a pregnancy is. Ultrasound can be pricey and hard to find. Some studies show that a protein in blood, called PAPP-A, may be another option. The authors took blood from and did ultrasounds for people who wanted an abortion. They measured how much PAPP-A was in the blood using two different methods. The level of PAPP-A in the blood did a good job of distinguishing pregnancies that were higher and lower than 71 days.

Transcriptomics-Based Subphenotyping of the Human Placenta Enabled by Weighted Correlation Network Analysis in Early-Onset Preeclampsia With and Without Fetal Growth Restriction.

BACKGROUND: Placental disorders contribute to pregnancy complications, including preeclampsia and fetal growth restriction (FGR), but debate regarding their specific pathobiology persists. Our objective was to apply transcriptomics with weighted gene correlation network analysis to further clarify the placental dysfunction in these conditions. METHODS: We performed RNA sequencing with weighted gene correlation network analysis using human placental samples (n=30), separated into villous tissue and decidua basalis, and clinically grouped as follows: (1) early-onset preeclampsia (EOPE)+FGR (n=7); (2) normotensive, nonanomalous preterm FGR (n=5); (2) EOPE without FGR (n=8); (4) spontaneous idiopathic preterm birth (n=5) matched for gestational age; and (5) uncomplicated term births (n=5). Our data was compared with RNA sequencing data sets from public databases (GSE114691, GSE148241, and PRJEB30656; n=130 samples). RESULTS: We identified 14 correlated gene modules in our specimens, of which most were significantly correlated with birthweight and maternal blood pressure. Of the 3 network modules consistently predictive of EOPE±FGR across data sets, we prioritized a coexpression gene group enriched for hypoxia-response and metabolic pathways for further investigation. Cluster analysis based on transcripts from this module and the glycolysis/gluconeogenesis metabolic pathway consistently distinguished a subset of EOPE±FGR samples with an expression signature suggesting modified tissue bioenergetics. We demonstrated that the expression ratios of LDHA/LDHB and PDK1/GOT1 could be used as surrogate indices for the larger panels of genes in identifying this subgroup. CONCLUSIONS: We provide novel evidence for a molecular subphenotype consistent with a glycolytic metabolic shift that occurs more frequently but not universally in placental specimens of EOPE±FGR.

Prevalence of and Risk Factors for Iron Deficiency in Twin and Singleton Newborns.

Iron deficiency (ID) in utero and in infancy can cause irreversible neurocognitive damage. Iron status is not routinely tested at birth, so the burden of neonatal ID in the United States is unknown. Infants born from twin or higher-order pregnancies may be at elevated risk of inadequate nutrient endowment at birth. The present study sought to compare the burden of neonatal ID in cord blood serum samples from twin (n = 54) and singleton pregnancies (n = 24). Iron status (serum ferritin (SF), soluble transferrin receptor (sTfR), hepcidin) and inflammation (C-reactive protein (CRP) and interleukin-6 (IL-6)) biomarker concentrations were measured by immunoassay. The prevalence of ID (SF < 76 ng/mL) among twins was 21% (23/108) and among singletons 20% (5/24). Gestational age at birth, maternal race and infant sex predicted SF levels. Maternal anemia (hemoglobin < 11 g/dL) was observed in 40% of mothers but was not associated with neonatal iron biomarkers. More research is needed to identify risk factors and regulatory mechanisms for inadequate fetal iron accrual to identify higher risk pregnancies and neonates for screening and intervention.

A Novel Role for Plasminogen Activator Inhibitor Type-2 as a Hypochlorite-Resistant Serine Protease Inhibitor and Holdase Chaperone.

Plasminogen activator inhibitor type-2 (PAI-2), a member of the serpin family, is dramatically upregulated during pregnancy and in response to inflammation. Although PAI-2 exists in glycosylated and non-glycosylated forms in vivo, the majority of in vitro studies of PAI-2 have exclusively involved the intracellular non-glycosylated form. This study shows that exposure to inflammation-associated hypochlorite induces the oligomerisation of PAI-2 via a mechanism involving dityrosine formation. Compared to plasminogen activator inhibitor type-1 (PAI-1), both forms of PAI-2 are more resistant to hypochlorite-induced inactivation of its protease inhibitory activity. Holdase-type extracellular chaperone activity plays a putative non-canonical role for PAI-2. Our data demonstrate that glycosylated PAI-2 more efficiently inhibits the aggregation of Alzheimer's disease and preeclampsia-associated amyloid beta peptide (Aß), compared to non-glycosylated PAI-2 in vitro. However, hypochlorite-induced modification of non-glycosylated PAI-2 dramatically enhances its holdase activity by promoting the formation of very high-molecular-mass chaperone-active PAI-2 oligomers. Both PAI-2 forms protect against Aß-induced cytotoxicity in the SH-SY5Y neuroblastoma cell line in vitro. In the villous placenta, PAI-2 is localised primarily to syncytiotrophoblast with wide interpersonal variation in women with preeclampsia and in gestational-age-matched controls. Although intracellular PAI-2 and Aß staining localised to different placental cell types, some PAI-2 co-localised with Aß in the extracellular plaque-like aggregated deposits abundant in preeclamptic placenta. Thus, PAI-2 potentially contributes to controlling aberrant fibrinolysis and the accumulation of misfolded proteins in states characterised by oxidative and proteostasis stress, such as in Alzheimer's disease and preeclampsia.

Endocan, a Soluble Marker of Endothelial Cell Activation Is a Molecular Marker of Disease Severity in Women with Preeclampsia.

Endocan is a proteoglycan secreted by activated endothelium that regulates angiogenesis via interaction with hepatocyte growth factor (HGF). We hypothesized that women diagnosed with preeclampsia (PE) and/or fetal growth restriction (FGR) have elevated circulating endocan concentrations in direct relationship with severity of clinical manifestations. Serum concentration of endocan and HGF were analyzed in 224 women grouped as healthy pregnant controls (P-CRL, n = 77), PE with severe features (sPE, n = 83), chronic hypertension (crHTN: n = 36), idiopathic FGR (n = 18), and healthy non-pregnant controls (NP-CRL, n = 7). Endocan and HGF measured by immunoassay were analyzed along with markers of inflammation, angiogenesis, and protein misfolding (urine congophilia). Endocan expression in the placenta and/or myometrium was studied by immunohistochemistry and real-time PCR. Compared to gestational age-matched P-CRL, women with early-onset sPE had higher circulating endocan concentrations. Among women with PE and/or FGR, endocan concentration correlated with soluble endoglin and urine congophilia but not with HGF or markers of inflammation or angiogenesis. In the placenta, endocan was expressed in villous and extravillous trophoblasts and endothelium. Intense endocan immunostaining was observed in plaque-like aggregations of sPE placentas complicated with FGR. In addition, thickened blood vessels in the myometrium of sPE patients stained positive for endocan. Women with early-onset sPE have elevated serum endocan likely reflecting chronic endothelial activation. Enhanced expression and/or deposition of endocan at the sites of placental injury and in remodeled maternal blood vessels supports a role for endocan in either vascular rescue or as a contributor to FGR and perhaps long-term cardiovascular morbidity.

Conformation-dependent anti-Aß monoclonal antibody signatures of disease status and severity in urine of women with preeclampsia.

Prior research has shown that urine of women with preeclampsia (PE) contains amyloid-like aggregates that are congophilic (exhibit affinity for the amyloidophilic dye Congo red) and immunoreactive with A11, a polyclonal serum against prefibrillar ß-amyloid oligomers, thereby supporting pathogenic similarity between PE and protein conformational disorders such as Alzheimer's and prion disease. The objective of this study was to interrogate PE urine using monoclonal antibodies with previously characterized A11-like epitopes. Over 100 conformation-dependent monoclonals were screened and three (mA11-09, mA11-89, and mA11-205) selected for further confirmation in 196 urine samples grouped as follows: severe features PE (sPE, n = 114), PE without severe features (mPE, n = 30), chronic hypertension (crHTN, n = 14) and normotensive pregnant control (P-CRL, n = 38). We showed that the selected conformation-specific monoclonals distinguished among patients with varying severities of PE from P-CRL and patients with crHTN. By use of latent class analysis (LCA) we identified three classes of subjects: Class 1 (n = 94) comprised patients whose urine was both congophilic and reactive with the monoclonals. These women were more likely diagnosed with early-onset sPE and had severe hypertension and proteinuria; Class 2 patients (n = 55) were negative for congophilia and against the antibodies. These were predominantly P-CRL and crHTN patients. Lastly, Class 3 patients (n = 48) were positive for urine congophilia, albeit at lower intensity, but negative for monoclonal immunoreactivities. These women were diagnosed primarily as mPE or late-onset sPE. Collectively, our study validates conformation-dependent Aß imunoreactivity of PE urine which in conjunction to urine congophilia may represent an additional indicator of disease severity.

Analytical Comparison of Pregnancy-Associated Plasma Protein-A (PAPP-A) Immunoassays for Biochemical Determination of Gestational Age.

BACKGROUND: Accurate pregnancy dating is critical for maternal and child health and for counseling on safe and effective abortion methods. While last menstrual period and first trimester ultrasound are often used together to determine gestational age (GA), they have limited accuracy and availability, respectively. Prior studies have shown that pregnancy-associated plasma protein-A (PAPP-A) increases exponentially during pregnancy and has the potential to serve as a biochemical marker of GA. We aimed to analyze the relationship between sonographically determined GA and serum PAPP-A concentration measured by different immunoassays and to derive cutoff levels informative for the 70 days GA commonly recommended limit for medical abortion in outpatient settings. METHODS: We compared technical characteristics of 4 commercially available PAPP-A immunoassays and tested 120 maternal serum samples (GA range: 34-231 days) along with contrived pool samples and traceable quality controls. These characteristics included area under the receiver operator characteristic (AUROC) plot, sensitivity and specificity based on cutoffs defined by the Youden Index, and likelihood ratios. RESULTS: All 4 immunoassays had sensitivities and specificities ≥80%, and AUROC values ranging from 0.948 to 0.968. Marked differences among absolute PAPP-A values were noted depending on immunoassay. PAPP-A cutoff values at 70 days GA for each individual immunoassay were established along with procedural recommendations that increase equivalence among immunoassays. CONCLUSIONS: Maternal serum PAPP-A levels correlated strongly with GA despite differences in immunoassay formats and absolute data output. Serum PAPP-A has biomarker potential for future development of a point-of-care test aimed at increasing access to medical abortion.

Molecular signatures of labor and nonlabor myometrium with parsimonious classification from 2 calcium transporter genes.

Clinical phenotyping of term and preterm labor is imprecise, and disagreement persists on categorization relative to underlying pathobiology, which remains poorly understood. We performed RNA sequencing (RNA-seq) of 31 specimens of human uterine myometrium from 10 term and 21 preterm cesarean deliveries with rich clinical context information. A molecular signature of 4814 transcripts stratified myometrial samples into quiescent (Q) and nonquiescent (NQ) phenotypes, independent of gestational age and incision site. Similar stratifications were achieved using expressed genes in Ca2+ signaling and TGF-ß pathways. For maximal parsimony, we evaluated the expression of just 2 Ca2+ transporter genes, ATP2B4 (encoding PMCA4) and ATP2A2 (coding for SERCA2), and we found that their ratio reliably distinguished NQ and Q specimens in the current study, and also in 2 publicly available RNA-seq data sets (GSE50599 and GSE80172), with an overall AUC of 0.94. Cross-validation of the ATP2B4/ATP2A2 ratio by quantitative PCR in an expanded cohort (by 11 additional specimens) achieved complete separation (AUC of 1.00) of NQ versus Q specimens. While providing additional insight into the associations between clinical features of term and preterm labor and myometrial gene expression, our study also offers a practical algorithm for unbiased classification of myometrial biopsies by their overall contractile program.

Amniotic Fluid Proteasome and Immunoproteasome in the Setting of Intra-Amniotic Infection, Inflammation, and Preterm Birth.

Preterm birth is an important determinant of neonatal morbidity and mortality and intra-amniotic infection (IAI) and inflammation play a causative role. The constitutive proteasome and immunoproteasome are key players in maintenance of proteostasis and their alteration outside pregnancy has been linked to pathogenesis of numerous inflammatory diseases. Our goal was to evaluate the levels, activities, and potential origin of amniotic fluid (AF) proteasome in women with preterm birth induced by infection and/or inflammation. Total proteasome and immunoproteasome concentrations were measured in AF retrieved by trans-abdominal amniocentesis from 155 pregnant women. Proteasome activities were measured with fluorogenic substrates targeting caspase-like (CAS-L), trypsin-like (TRY-L), or chymotrypsin-like (CHE-L) lytic activities. We found that IAI significantly upregulated AF concentrations of total proteasome and of the immunoproteasome (P<0.001 for both) with no differences based on gestational age. Based on substrate preference and profile of pharmacologic inhibition, we identified the CHE-L activity of the immunoproteasome as the primary lytic activity upregulated in AF of pregnancies complicated by IAI. When compared with matched maternal blood and cord blood, proteasome activity was by far the highest in AF and this was further elevated in IAI. Western blot confirmed ß5 (PSMB5) and ß5i (PSMB8) subunits of the constitutive proteasome and immunoproteasome are present in AF and IHC staining of fetal membranes pointed to chorio-decidua as a potential source. In conclusion, IAI is associated with increased AF immunoproteasome activity that by analogy with other inflammatory diseases may generate antigenic oligopeptides and may play a role in triggering preterm birth.

Antenatal N-acetylcysteine to improve outcomes of premature infants with intra-amniotic infection and inflammation (Triple I): randomized clinical trial.

BACKGROUND: Intrauterine infection and/or inflammation (Triple I) is an important cause of preterm birth (PTB) and adverse newborn outcomes. N-acetylcysteine (NAC) is a Food and Drug Administration (FDA)-approved drug safely administered to pregnant women with acetaminophen toxicity. METHODS: We conducted a single-center, quadruple-blind, placebo-controlled trial of pregnant women with impending PTB due to confirmed Triple I. Participants (n = 67) were randomized to an intravenous infusion of NAC or placebo mimicking the FDA-approved regimen. Outcomes included clinical measures and mechanistic biomarkers. RESULTS: Newborns exposed to NAC (n = 33) had significantly improved status at birth and required less intensive resuscitation compared to placebo (n = 34). Fewer NAC-exposed newborns developed two or more prematurity-related severe morbidities [NAC: 21% vs. placebo: 47%, relative risk, 0.45; 95% confidence interval (CI) 0.21-0.95] with the strongest protection afforded against bronchopulmonary dysplasia (BPD, NAC: 3% vs. placebo: 32%, relative risk, 0.10; 95% CI: 0.01-0.73). These effects were independent of gestational age, birth weight, sex, or race. Umbilical cord plasma NAC concentration correlated directly with cysteine, but not with plasma or whole blood glutathione. NAC reduced the placental expression of histone deacetylase-2, suggesting that epigenetic mechanisms may be involved. CONCLUSIONS: These data provide support for larger studies of intrapartum NAC to reduce prematurity-related morbidity. IMPACT: In this randomized clinical trial of 65 women and their infants, maternal intravenous NAC employing the FDA-approved dosing protocol resulted in lower composite neonatal morbidity independent of gestational age, race, sex, and birthweight. Administration of NAC in amniocentesis-confirmed Triple I resulted in a remarkably lower incidence of BPD. As prior studies have not shown a benefit of postnatal NAC in ventilated infants, our trial highlights the critical antenatal timing of NAC administration. Repurposing of NAC for intrapartum administration should be explored in larger clinical trials as a strategy to improve prematurity-related outcomes and decrease the incidence of BPD.

Connecting the dots on vertical transmission of SARS-CoV-2 using protein-protein interaction network analysis - Potential roles of placental ACE2 and ENDOU.

We conducted a protein-protein interaction (PPI) network study searching for proteins relevant to pregnancy-associated COVID-19 in pregnancy complicated with severe preeclampsia (sPE) and intra-amniotic infection and/or inflammation (Triple-I). PPI networks from sPE and Triple-I were intersected with the PPI network from coronavirus infection. Common proteins included the SARS-CoV-2 entry receptor ACE2 and ENDOU, a placental endoribonuclease homologous to Nsp15, a protein produced by the virus to escape host immunity. Remarkably, placental ENDOU mRNA expression far exceeded that of ACE2. Immunohistochemistry confirmed ENDOU localization at the hemochorial maternal-fetal interface. Investigation of ENDOU's relevance to vertical transmission of SARS-CoV-2 is further warranted.

Electrophysiological Maturation of Cerebral Organoids Correlates with Dynamic Morphological and Cellular Development.

Cerebral organoids (COs) are rapidly accelerating the rate of translational neuroscience based on their potential to model complex features of the developing human brain. Several studies have examined the electrophysiological and neural network features of COs; however, no study has comprehensively investigated the developmental trajectory of electrophysiological properties in whole-brain COs and correlated these properties with developmentally linked morphological and cellular features. Here, we profiled the neuroelectrical activities of COs over the span of 5 months with a multi-electrode array platform and observed the emergence and maturation of several electrophysiologic properties, including rapid firing rates and network bursting events. To complement these analyses, we characterized the complex molecular and cellular development that gives rise to these mature neuroelectrical properties with immunohistochemical and single-cell transcriptomic analyses. This integrated approach highlights the value of COs as an emerging model system of human brain development and neurological disease.

Acute Glucose Load, Inflammation, Oxidative Stress, Nonenzymatic Glycation, and Screening for Gestational Diabetes.

AIMS: To investigate if oral glucose tolerance test (OGTT) associates with changes in maternal symptoms (ie, flushing, sweating), blood nonenzymatic advanced glycation end products (AGE), acute-phase reactive inflammatory markers, and oxidative stress. METHODS: Prospective case-control study of patients screened for gestational diabetes mellitus (GDM). One hundred nonfasting, second-trimester consecutive pregnant women allocated to either 50 g OGTT or water. Five women who had a 3-hour fasting 100 g OGTT also enrolled. Maternal serum glucose, AGE, soluble receptor for AGE (sRAGE), interleukin (IL)-6, and C-reactive protein (CRP) were immunoassayed. Total radical-trapping antioxidant parameter (TRAP) estimated with antioxidant capacityperoxyl assay. Data corrected for gestational age and maternal body mass index. RESULTS: During 50 g OGTT there was a decrease in systolic blood pressure not accompanied by the onset of adverse clinical symptoms. There was a decrease in serum glucose levels 1 hour after water (P = .019) but not glucose ingestion. Serum CRP (P = .001) but not IL-6 was increased. The AGE, sRAGE, and TRAP levels remained unchanged. Similar results were seen during 100 g OGTT, except serum glucose was significantly elevated after 1 hour. CONCLUSION: Results suggest screening tools for gestational diabetes are safe and clinically well tolerated during pregnancy. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov NCT03029546.

Fetal Myocardial Function as Assessed by N-Terminal Fragment Brain Natriuretic Protein in Premature Fetuses Exposed to Intra-amniotic Inflammation.

OBJECTIVE: This study aimed to determine the relationship between fetal exposure to intra-amniotic infection/inflammation (IAI) and fetal heart ventricular function as assessed by circulatory levels of N-terminal fragment brain natriuretic protein (NT-proBNP) and the Tei index. STUDY DESIGN: We analyzed 70 samples of paired amniotic fluid (AF) and cord blood retrieved from mothers who delivered preterm at <34 weeks as follows: Yes-IAI (n = 36) and No-IAI (n = 34). IAI was diagnosed by amniocentesis and AF mass spectrometry. Fetal exposure to inflammation was determined through the evaluation of cord blood haptoglobin (Hp) switch-on status and level, and interleukin (IL)-6 levels by Western blotting and enzyme-linked immunosorbent assay, respectively. Fetal heart function was assessed by cord blood NT-proBNP immunoassay and fetal echocardiogram (Tei index). RESULTS: IAI was characterized by significantly higher levels of AF (p < 0.001) and umbilical cord IL-6 (p = 0.004). Cord blood Hp levels and frequency of switch-on status were higher in fetuses exposed to IAI (p < 0.001, both). Fetuses exposed to IAI did not have higher levels of NT-proBNP. Following correction for gestational age and race, neither cord blood NT-proBNP nor the Tei index was significantly different in fetuses with Hp switched-on status (p > 0.05, both). CONCLUSION: Fetal myocardial left ventricular function does not seem to be significantly impaired in fetuses born alive due to IAI if delivery of the fetus occurs immediately following the diagnosis of IAI.

Congo Red Dot Paper Test for Antenatal Triage and Rapid Identification of Preeclampsia.

BACKGROUND: Proteins in the urine of women with preeclampsia (PE) bind Congo Red dye (urine congophilia). We sought to determine the diagnostic performance of a paper-based point-of-care test detecting urine congophilia for rapid triage and diagnosis of PE. METHODS: Prospective cohort study conducted in 346 consecutive pregnant women evaluated for PE in the Labour and Delivery triage unit at our institution. The Congo Red Dot (CRD) Paper Test (index test) was performed on fresh urine samples. The CRD Paper Test results were compared to an expert adjudicated diagnosis in each case. The accuracy of the CRD Paper Test was also compared to urine and serum analytes (placental growth factor and soluble fms-like tyrosine kinase-1) previously proposed as diagnostic aids for PE. FINDINGS: During the first triage visit, 32% (112/346) of women received a clinical diagnosis of PE. Yet, 63% (217/346) were admitted for in-patient diagnostic work-up or delivery. The CRD Paper Test was positive in 25% (86/346) of the cases. Adjudication confirmed PE in 28% (96/346) of all cases. The CRD Paper Test outperformed measured serum and urine markers (80·2% sensitivity, 89·2% specificity, 92·1% negative predictive value, 86·7% accuracy). The pre-test, positive and negative post-test probabilities were 27·7%, 74·0%, and 8·0%, respectively. Of women who were discharged undelivered, 38% (133/346) had at least one additional triage visit and the interval between the last negative and first positive CRD Paper Test was 12 (interquartile range, [5-34]) days. INTERPRETATION: The CRD Paper Test is a simple, non-invasive, "sample-in/answer-out" point-of-care clinical tool for rapid identification of PE. FUNDING: Saving Lives at Birth Program and NICHD.

Proteasome Levels and Activity in Pregnancies Complicated by Severe Preeclampsia and Hemolysis, Elevated Liver Enzymes, and Thrombocytopenia (HELLP) Syndrome.

Excessive accumulation of misfolded proteins was recently demonstrated in preeclampsia. We examined levels and activity of circulatory proteasome and immunoproteasome (inflammatory subtype) in preeclampsia and hemolysis, elevated liver enzymes, and thrombocytopenia (HELLP) syndrome. We analyzed samples from women with hypertensive pregnancy disorders (n=115), including preeclampsia with severe features (sPE) and HELLP syndrome, and normotensive controls (n=45). Plasma proteasome and immunoproteasome immunoreactivity were determined by quantifying the α-subunit of the 20S core and ß5i (proteasome subunit beta 8 [PSMB8]), respectively. Plasma proteasome activity was analyzed with fluorogenic substrates. MG132, lactacystin, and ONX0914 were used to inhibit the circulating proteasome and immunoproteasome, respectively. Plasma cytokine profiles were evaluated by multiplex immunoassay. Placental expression of ß5 (constitutive proteasome) and ß5i (immunoproteasome) was interrogated by immunohistochemistry. Women with sPE had increased plasma 20S levels ( P<0.001) and elevated lytic activities (chymotrypsin-like 7-fold, caspase-like 4.2-fold, trypsin-like 2.2-fold; P <0.001 for all) compared with pregnant controls. Women with features of HELLP displayed the highest plasma proteasome levels and activity, which correlated with decreased IFN-γ (interferon-γ), and increased IL (interleukin)-8 and IL-10. In sPE and HELLP, chymotrypsin-like activity was suppressed by proteasome inhibitors including ONX0914. Compared with gestational age-matched controls, sPE placentas harbored increased ß5 and ß5i immunostaining in trophoblasts. ß5i signal was elevated in HELLP with predominant staining in villous core, extravillous trophoblasts in placental islands, and extracellular vesicles in intervillous spaces. Pregnancy represents a state of increased proteostatic stress. sPE and HELLP were characterized by significant upregulation in circulating levels and lytic activity of the proteasome that was partially explained by placental immunoproteasome upregulation.

Acute Glucose Load, Inflammation, Oxidative Stress, Nonenzymatic Glycation, and Screening for Gestational Diabetes.

AIMS:: To investigate if oral glucose tolerance test (OGTT) associates with changes in maternal symptoms (ie, flushing, sweating), blood nonenzymatic advanced glycation end products (AGE), acute-phase reactive inflammatory markers, and oxidative stress. METHODS:: Prospective case-control study of patients screened for gestational diabetes mellitus (GDM). One hundred nonfasting, second-trimester consecutive pregnant women allocated to either 50 g OGTT or water. Five women who had a 3-hour fasting 100 g OGTT also enrolled. Maternal serum glucose, AGE, soluble receptor for AGE (sRAGE), interleukin (IL)-6, and C-reactive protein (CRP) were immunoassayed. Total radical-trapping antioxidant parameter (TRAP) estimated with antioxidant capacity-peroxyl assay. Data corrected for gestational age and maternal body mass index. RESULTS:: During 50 g OGTT there was a decrease in systolic blood pressure not accompanied by the onset of adverse clinical symptoms. There was a decrease in serum glucose levels 1 hour after water ( P = .019) but not glucose ingestion. Serum CRP ( P = .001) but not IL-6 was increased. The AGE, sRAGE, and TRAP levels remained unchanged. Similar results were seen during 100 g OGTT, except serum glucose was significantly elevated after 1 hour. CONCLUSION:: Results suggest screening tools for gestational diabetes are safe and clinically well tolerated during pregnancy. Clinical Trial Registration: ClinicalTrials.gov NCT03029546.

Human pregnancy zone protein stabilizes misfolded proteins including preeclampsia- and Alzheimer's-associated amyloid beta peptide.

Protein misfolding underlies the pathology of a large number of human disorders, many of which are age-related. An exception to this is preeclampsia, a leading cause of pregnancy-associated morbidity and mortality in which misfolded proteins accumulate in body fluids and the placenta. We demonstrate that pregnancy zone protein (PZP), which is dramatically elevated in maternal plasma during pregnancy, efficiently inhibits in vitro the aggregation of misfolded proteins, including the amyloid beta peptide (Aß) that is implicated in preeclampsia as well as with Alzheimer's disease. The mechanism by which this inhibition occurs involves the formation of stable complexes between PZP and monomeric Aß or small soluble Aß oligomers formed early in the aggregation pathway. The chaperone activity of PZP is more efficient than that of the closely related protein alpha-2-macroglobulin (α2M), although the chaperone activity of α2M is enhanced by inducing its dissociation into PZP-like dimers. By immunohistochemistry analysis, PZP is found primarily in extravillous trophoblasts in the placenta. In severe preeclampsia, PZP-positive extravillous trophoblasts are adjacent to extracellular plaques containing Aß, but PZP is not abundant within extracellular plaques. Our data support the conclusion that the up-regulation of PZP during pregnancy represents a major maternal adaptation that helps to maintain extracellular proteostasis during gestation in humans. We propose that overwhelming or disrupting the chaperone function of PZP could underlie the accumulation of misfolded proteins in vivo. Attempts to characterize extracellular proteostasis in pregnancy will potentially have broad-reaching significance for understanding disease-related protein misfolding.

Toll-like receptor 9, maternal cell-free DNA and myometrial cell response to CpG oligodeoxynucleotide stimulation.

PROBLEM: Among mechanisms triggering onset of parturition, it has been recently postulated that Toll-Like Receptor (TLR)9 engagement by cell-free DNA (cfDNA) triggers inflammation, myometrial contractions, and labor in absence of infection. The current study evaluated whether direct (myometrial) or indirect (decidual) TLR9 engagement enhances human myometrial contractility. METHOD OF STUDY: Toll-like receptor 9 expression and cellular localization were surveyed by immunohistochemistry of placenta, fetal membranes, and myometrium in term (gestational age [GA]: >37 weeks) labor (TL, n = 7) or term non-labor (TNL, n = 7) tissues. Non-pregnant myometrium (n = 4) served as reference. TLR9 mRNA expression relative to other TLRs was evaluated through the mining of an RNA-seq dataset and confirmed by RT-PCR. Immortalized human myometrial cells (hTERT-HM) were treated with incremental concentrations of TLR9 agonist ODN2395, TNF-α, or LPS. Secreted cytokines were quantified by multiplex immunoassay, and contractility was assessed by an in-gel cell contraction assay (n = 9). Induction of hTERT-HM contractility was also evaluated indirectly following exposure to conditioned media from primary term decidual cells (n = 4) previously stimulated with ODN2395. RESULTS: Toll-like receptor 9 immunostaining in placenta and amniochorion was strongest in decidual cells, but unrelated to labor. TLR9 staining intensity was significantly decreased in TL compared with TNL myometrium (P = 0.002). Although total cfDNA in maternal circulation increased in TL (P = 0.025 vs TNL), difference in cffDNA was non-significant. Myometrial TLR9 mRNA levels were unaffected by contractile status and far less abundant than other pro-inflammatory TLRs. hTERT-HM contractility was enhanced by LPS (P = 0.002) and TNF-α (P = 0.003), but not by ODN2395 (P = 0.345) or supernatant of TLR9-stimulated decidual cells. CONCLUSION: Myometrial and decidual TLR9 are unlikely to directly regulate human parturition.

Tenascin-X in amniotic fluid and reproductive tissues of pregnancies complicated by infection and preterm prelabor rupture of membranes†.

Preterm prelabor rupture of membranes (PPROM), which can precede or follow intra-amniotic infection/inflammation (IAI), is a poorly understood pregnancy complication. Tenascin-X (TNX) is a connective tissue extracellular matrix protein that regulates fibrillogenesis of collagens I, III, and V. Our goal was to investigate the presence and level of soluble TNX (sTNX) in amniotic fluid (AF) and TNX expression in reproductive tissues of pregnancies complicated by PPROM and IAI. We prospectively recruited 334 women pregnant with singletons who had a clinically indicated amniocentesis for genetic karyotyping, lung maturity testing, or rule-out IAI in the presence or absence of PPROM. We quantified TNX expression in fetal membranes, myometrium, cervix, and placenta using immunological methods and qRT-PCR. In pregnancies with normal outcomes, AF sTNX levels were GA-regulated with lower levels toward term. IAI significantly upregulated AF sTNX levels independent of membrane status. AF sTNX levels inversely correlated with fetal membranes tenascin XB (TNXB) mRNA level, which was significantly downregulated by IAI. Western blotting identified characteristic ∼75 and ∼140 kDa sTNX forms in both AF and fetal membranes. Fetal membranes, placenta, and cervix constitutively express TNX with the highest abundance in the amnion. Amnion TNX richness is significantly lost in the setting of IAI. Our results suggest that fetal membranes may be a source of AF sTNX whereby protein and mRNA expression seem to be significantly impacted by inflammation independent of fetal membrane status. A more thorough understanding of TNX changes may be valuable for understanding spontaneous PPROM and to potentially develop therapeutic targets.