Identification of circRNA Expression Profile and Potential Systemic Immune Imbalance Modulation in Premature Rupture of Membranes.

Premature rupture of membrane (PROM) refers to the rupture of membranes before the onset of labor which increases the risk of perinatal morbidity and mortality. Recently, circular RNAs (circRNAs) have emerged as promising regulators of diverse diseases. However, the circRNA expression profiles and potential circRNA-miRNA-mRNA regulatory mechanisms in PROM remain enigmatic. In this study, we displayed the expression profiles of circRNAs and mRNAs in plasma and fetal membranes of PROM and normal control (NC) groups based on circRNA microarray, the Gene Expression Omnibus database, and NCBI's Sequence Read Archive. A total of 1,459 differentially expressed circRNAs (DECs) in PROM were identified, with 406 upregulated and 1,053 downregulated. Then, we constructed the circRNA-miRNA-mRNA network in PROM, encompassing 22 circRNA-miRNA pairs and 128 miRNA-mRNA pairs. Based on the analysis of gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and gene set enrichment analysis (GSEA), DECs were implicated in immune-related pathways, with certain alterations persisting even postpartum. Notably, 11 host genes shared by DECs of fetal membrane tissue and prenatal plasma in PROM were significantly implicated in inflammatory processes and extracellular matrix regulation. Our results suggest that structurally stable circRNAs may predispose to PROM by mediating systemic immune imbalances, including peripheral leukocyte disorganization, local immune imbalance at the maternal-fetal interface, and local collagen disruption. This is the first time to decipher a landscape on circRNAs of PROM, reveals the pathogenic cause of PROM from the perspective of circRNA, and opens up a new direction for the diagnosis and treatment of PROM.

mRNA and Protein Expression in Human Fetal Membrane Cells: Potential Biomarkers for Preterm Prelabor Rupture of the Fetal Membranes?

Clinically, unique markers in fetal membrane cells may contribute to the search for biomarkers for preterm prelabor rupture of the fetal membranes (pPROM) in maternal blood. pPROM is associated with overwhelming inflammation and premature cellular senescence causing "biological microfractures" of the fetal membranes. We hypothesize that these pathological processes are associated with the shedding of fetal membrane cells into the maternal circulation. The aim of this study was to identify markers expressed exclusively in fetal membrane cells to facilitate their isolation, characterization, and determination of biomarker potential in maternal blood. We have (1), by their transcriptomic profile, identified markers that are upregulated in amnion and chorion tissue compared to maternal white blood cells, and (2), by immunohistochemistry, confirmed the localization of the differentially expressed proteins in fetal membranes, placenta, and the placental bed of the uterus. RNA sequencing revealed 31 transcripts in the amnion and 42 transcripts in the chorion that were upregulated. Among these, 22 proteins were evaluated by immunohistochemistry. All but two transcripts were expressed both on mRNA and protein level in at least one fetal membrane cell type. Among these remaining 20 proteins, 9 proteins were not significantly expressed in the villous and extravillous trophoblasts of the placenta.

Novel genetic variants linked to prelabor rupture of membranes among Chinese pregnant women.

INTRODUCTION: The etiology of prelabor rupture of membranes (PROM), either preterm or term PROM (PPROM or TPROM), remains largely unknown. This study aimed to investigate the association between maternal genetic variants (GVs) and PROM and further establish a GV-based prediction model for PROM. METHODS: In this case-cohort study (n = 1166), Chinese pregnant women with PPROM (n = 51), TPROM (n = 283) and controls (n = 832) were enrolled. A weighted Cox model was applied to identify the GVs (single nucleotide polymorphisms [SNPs], insertions/deletions, and copy number variants) associated with either PPROM or TPROM. Gene set enrichment analysis (GSEA) was to explore the mechanisms. The suggestively significant GVs were applied to establish a random forest (RF) model. RESULTS: PTPRT variants (rs117950601, P = 4.37 × 10-9; rs147178603, P = 8.98 × 10-9) and SNRNP40 variant (rs117573344, P = 2.13 × 10-8) were associated with PPROM. STXBP5L variant (rs10511405, P = 4.66 × 10-8) was associated with TPROM. GSEA results showed that genes associated with PPROM were enriched in cell adhesion, and TPROM in ascorbate and glucuronidation metabolism. The area under the receiver operating characteristic curve of SNP-based RF model for PPROM was 0.961, with a sensitivity of 100.0% and specificity of 83.3%. DISCUSSION: Maternal GVs in PTPRT and SNRNP40 were associated with PPROM, and GV in STXBP5L was associated with TPROM. Cell adhesion participated in PPROM, while ascorbate and glucuronidation metabolism contributed in TPROM. The PPROM might be well predicted using the SNP-based RF model.

Circulating Extracellular Vesicles microRNAs Are Altered in Women Undergoing Preterm Birth.

Preterm labor (PTL) and preterm premature rupture of membranes (PPROM) lead to high perinatal morbidity/mortality rates worldwide. Small extracellular vesicles (sEV) act in cell communication and contain microRNAs that may contribute to the pathogenesis of these complications. We aimed to compare the expression, in sEV from peripheral blood, of miRNAs between term and preterm pregnancies. This cross-sectional study included women who underwent PTL, PPROM, and term pregnancies, examined at the Botucatu Medical School Hospital, SP, Brazil. sEV were isolated from plasma. Western blot used to detect exosomal protein CD63 and nanoparticle tracking analysis were performed. The expression of 800 miRNAs was assessed by the nCounter Humanv3 miRNA Assay (NanoString). The miRNA expression and relative risk were determined. Samples from 31 women-15 preterm and 16 term-were included. miR-612 expression was increased in the preterm groups. miR-612 has been shown to increase apoptosis in tumor cells and to regulate the nuclear factor κB inflammatory pathway, processes involved in PTL/PPROM pathogenesis. miR-1253, miR-1283, miR378e, and miR-579-3p, all associated with cellular senescence, were downregulated in PPROM compared with term pregnancies. We conclude that miRNAs from circulating sEV are differentially expressed between term and preterm pregnancies and modulate genes in pathways that are relevant to PTL/PPROM pathogenesis.

MicroRNA analysis in maternal blood of pregnancies with preterm premature rupture of membranes reveals a distinct expression profile.

OBJECTIVE: To determine the expression profile of microRNAs in the peripheral blood of pregnant women with preterm premature rupture of membranes (PPROM) compared to that of healthy pregnant women. STUDY DESIGN: This was a pilot study with case-control design in pregnant patients enrolled between January 2017 and June 2019. Patients with healthy pregnancies and those affected by PPROM between 20- and 33+6 weeks of gestation were matched by gestational age and selected for inclusion to the study. Patients were excluded for multiple gestation and presence of a major obstetrical complication such as preeclampsia, diabetes, fetal growth restriction and stillbirth. A total of ten (n = 10) controls and ten (n = 10) patients with PPROM were enrolled in the study. Specimens were obtained before administration of betamethasone or intravenous antibiotics. MicroRNA expression was analyzed for 800 microRNAs in each sample using the NanoString nCounter Expression Assay. Differential expression was calculated after normalization and log2- transformation using the false discovery rate (FDR) method at an alpha level of 5%. RESULTS: Demographic characteristics were similar between the two groups. Of the 800 miRNAs analyzed, 116 were differentially expressed after normalization. However, only four reached FDR-adjusted statistical significance. Pregnancies affected by PPROM were characterized by upregulation of miR-199a-5p, miR-130a-3p and miR-26a-5p and downregulation of miR-513b-5p (FDR adjusted p-values <0.05). The differentially expressed microRNAs participate in pathways associated with altered collagen and matrix metalloprotease expression in the extracellular matrix. CONCLUSION: Patients with PPROM have a distinct peripheral blood microRNA profile compared to healthy pregnancies as measured by the NanoString Expression Assay.

The Implication of Aquaporin-9 in the Pathogenesis of Preterm Premature Rupture of Membranes.

OBJECTIVE: This study aimed to detect aquaporin-9 (AQP9) concentrations in the serum of patients with preterm premature rupture of membranes (PPROM) and compare them with the healthy control group with intact membranes. MATERIAL AND METHODS: We conducted this prospective case-control study from March 2021 to August 2021. Of the 80 pregnant patients included in the study, we enrolled 42 singleton pregnant patients with PPROM as the study group and 43 healthy gestational age-, and body mass index (BMI)-matched healthy pregnant women with intact fetal membranes as the control group. We compared demographic and clinical characteristics, complete blood count and biochemical parameters, and serum AQP9 concentrations of the participants. We constructed an ROC curve to illustrate the sensitivity and specificity performance characteristics of AQP9 and calculated a cutoff value by using the Youden index. RESULTS: Maternal serum AQP-9 concentrations were significantly higher in patients with PPROM (804.46±195.63 pg/mL) compared to the healthy pregnant women in the control group (505.97±68.89 pg/mL, p<0.001). When we examine the area under the ROC curve (AUC), the AQP-9 value can be reflected as a statistically significant parameter for diagnosing PPROM. According to the Youden index, a 654.78 pg/mL cut-off value of AQP-9 can be utilized to diagnose PPROM with 80.5% sensitivity and 100% specificity. CONCLUSION: Maternal serum AQP9 concentrations were significantly higher in PPROM patients than healthy pregnant women with an intact membrane. We suggest that AQP9 might be an essential biomarker of the inflammatory process and energy homeostasis in PPROM.

Genome and transcriptome profiling of spontaneous preterm birth phenotypes.

Preterm birth (PTB) occurs before 37 weeks of gestation. Risk factors include genetics and infection/inflammation. Different mechanisms have been reported for spontaneous preterm birth (SPTB) and preterm birth following preterm premature rupture of membranes (PPROM). This study aimed to identify early pregnancy biomarkers of SPTB and PPROM from the maternal genome and transcriptome. Pregnant women were recruited at the Liverpool Women's Hospital. Pregnancy outcomes were categorised as SPTB, PPROM (≤ 34 weeks gestation, n = 53), high-risk term (HTERM, ≥ 37 weeks, n = 126) or low-risk (no history of SPTB/PPROM) term (LTERM, ≥ 39 weeks, n = 188). Blood samples were collected at 16 and 20 weeks gestation from which, genome (UK Biobank Axiom array) and transcriptome (Clariom D Human assay) data were acquired. PLINK and R were used to perform genetic association and differential expression analyses and expression quantitative trait loci (eQTL) mapping. Several significant molecular signatures were identified across the analyses in preterm cases. Genome-wide significant SNP rs14675645 (ASTN1) was associated with SPTB whereas microRNA-142 transcript and PPARG1-FOXP3 gene set were associated with PPROM at week 20 of gestation and is related to inflammation and immune response. This study has determined genomic and transcriptomic candidate biomarkers of SPTB and PPROM that require validation in diverse populations.

Labour classified by cervical dilatation & fetal membrane rupture demonstrates differential impact on RNA-seq data for human myometrium tissues.

High throughput sequencing has previously identified differentially expressed genes (DEGs) and enriched signalling networks in human myometrium for term (≥37 weeks) gestation labour, when defined as a singular state of activity at comparison to the non-labouring state. However, transcriptome changes that occur during transition from early to established labour (defined as ≤3 and >3 cm cervical dilatation, respectively) and potentially altered by fetal membrane rupture (ROM), when adapting from onset to completion of childbirth, remained to be defined. In the present study, we assessed whether differences for these two clinically observable factors of labour are associated with different myometrial transcriptome profiles. Analysis of our tissue ('bulk') RNA-seq data (NCBI Gene Expression Omnibus: GSE80172) with classification of labour into four groups, each compared to the same non-labour group, identified more DEGs for early than established labour; ROM was the strongest up-regulator of DEGs. We propose that lower DEGs frequency for early labour and/or ROM negative myometrium was attributed to bulk RNA-seq limitations associated with tissue heterogeneity, as well as the possibility that processes other than gene transcription are of more importance at labour onset. Integrative analysis with future data from additional samples, which have at least equivalent refined clinical classification for labour status, and alternative omics approaches will help to explain what truly contributes to transcriptomic changes that are critical for labour onset. Lastly, we identified five DEGs common to all labour groupings; two of which (AREG and PER3) were validated by qPCR and not differentially expressed in placenta and choriodecidua.

Single Nucleotide Polymorphisms from CSF2, FLT1, TFPI and TLR9 Genes Are Associated with Prelabor Rupture of Membranes.

A prelabor rupture of membranes (PROM) and its subtypes, preterm PROM (pPROM) and term PROM (tPROM), are associated with disturbances in the hemostatic system and angiogenesis. This study was designed to demonstrate the role of single nucleotide polymorphisms (SNPs), localized in CSF2 (rs25881), FLT1 (rs722503), TFPI (C-399T) and TLR9 (rs352140) genes, in PROM. A population of 360 women with singleton pregnancy consisted of 180 PROM cases and 180 healthy controls. A single-SNP analysis showed a similar distribution of genotypes in the studied polymorphisms between the PROM or the pPROM women and the healthy controls. Double-SNP TT variants for CSF2 and FLT1 polymorphisms, CC variants for TLR9 and TFPI SNPs, TTC for CSF2, FLT1 and TLR9 polymorphisms, TTT for FLT1, TLR9 and TFPI SNPs and CCCC and TTTC complex variants for all tested SNPs correlated with an increased risk of PROM after adjusting for APTT, PLT parameters and/or pregnancy disorders. The TCT variants for the CSF2, FLT1 and TLR9 SNPs and the CCTC for the CSF2, FLT1, TLR9 and TFPI polymorphisms correlated with a reduced risk of PROM when corrected by PLT and APTT, respectively. We concluded that the polymorphisms of genes, involved in hemostasis and angiogenesis, contributed to PROM.

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.

A History of Preterm Delivery Is Associated with Aberrant Postpartal MicroRNA Expression Profiles in Mothers with an Absence of Other Pregnancy-Related Complications.

This prospective cross-sectional case-control study investigated the postpartal gene expression of microRNAs associated with diabetes/cardiovascular/cerebrovascular diseases in the peripheral white blood cells of women with anamnesis of preterm prelabor rupture of membranes (n = 58), spontaneous preterm birth (n = 55), and term delivery (n = 89) by a quantitative reverse transcription polymerase chain reaction. After pregnancies complicated by preterm prelabor rupture of membranes or spontaneous preterm birth, mothers showed diverse expression profiles for 25 out of 29 tested microRNAs (miR-1-3p, miR-16-5p, miR-17-5p, miR-20a-5p, miR-20b-5p, miR-21-5p, miR-23a-3p, miR-24-3p, miR-26a-5p, miR-29a-3p, miR-100-5p, miR-103a-3p, miR-125b-5p, miR-126-3p, miR-130b-3p, miR-133a-3p, miR-143-3p, miR-145-5p, miR-146a-5p, miR-181a-5p, miR-195-5p, miR-199a-5p, miR-221-3p, miR-499a-5p, and miR-574-3p). The earliest gestational ages at delivery and the lowest birth weights of newborns were associated with the highest postpartal levels of the previously mentioned microRNAs in maternal peripheral white blood cells. Administration of tocolytic drugs in order to prolong pregnancy, used in order to administer and complete a full course of antenatal corticosteroids, was associated with alterations in postpartal microRNA expression profiles to a lesser extent than in women with imminent delivery, where there was insufficient time for administration of tocolytics and antenatal corticosteroids. Overall, mothers who did not receive tocolytic therapy (miR-24-3p and miR-146a-5p) and mothers who did not receive corticosteroid therapy (miR-1-3p, miR-100-5p, and miR-143-3p) had increased or showed a trend toward increased postpartal microRNA expression when compared with mothers given tocolytic and corticosteroid therapy. In addition, mothers with serum C-reactive protein levels above 20 mg/L, who experienced preterm labour, showed a trend toward increased postpartal expression profiles of miR-143-3p and miR-199a-5p when compared with mothers with normal serum C-reactive protein levels. On the other hand, the occurrence of maternal leukocytosis, the presence of intra-amniotic inflammation (higher levels of interleukin 6 in the amniotic fluid), and the administration of antibiotics at the time of preterm delivery had no impact on postpartal microRNA expression profiles in mothers with a history of preterm delivery. Likewise, the condition of the newborns at the moment of birth, determined by Apgar scores at 5 and 10 min and the pH of cord arterial blood, had no influence on the postpartal expression profiles of mothers with a history of preterm delivery. These findings may contribute to explaining the increased cardiovascular risk in mothers with anamnesis of preterm delivery, and the greater increase of maternal cardiovascular risk with the decrease of gestational age at delivery. Women with preterm delivery in their anamnesis represent a high-risk group with special needs on a long-term basis, with a need to apply preventive and therapeutic interventions as early as possible.

Unfolding the pathophysiology of congenital thrombotic thrombocytopenic purpura in pregnancy: lessons from a cluster of familial cases.

BACKGROUND: Thrombotic thrombocytopenic purpura (TTP), a rare, potentially life-threatening thrombotic microangiopathy, manifests either as congenital TTP or acquired forms. It is caused by the absence or severe depletion of a disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS13) protease, leading to the accumulation of ultra large von Willebrand factor multimers as well as extensive platelet adhesion and clumping, which can ultimately cause severe secondary end-organ damage. Pregnancy can provoke or exacerbate TTP, leading to maternal and fetal complications. OBJECTIVE: In this report, we focused on pregnancy outcomes in a recently recognized cohort of congenital TTP patients of Bedouin Arab descent in southern Israel who were all homozygous for a novel c.3772delA variant of the ADAMTS13 gene, leading to the clinical manifestations of TTP largely during pregnancy. STUDY DESIGN: All patients presented in this study belong to 2 closely related families of Arab Bedouin descent and were found to be homozygous for a novel ADAMTS13-c.3772delA variant. The cohort consisted of 19 females; 16 of them had congenital TTP and had been pregnant and were thus included. Patient data were collected from electronic medical records. RESULTS: Of note, 13 women from our cohort, who delivered 14 fetuses (owing to 1 twin pregnancy), were diagnosed with congenital TTP following complicated pregnancies, which included recurrent pregnancy loss, stillbirth, early onset preeclampsia (both mild and severe), hemolysis, elevated liver enzymes and low platelet count syndrome, intrauterine growth restriction with abnormal Doppler flow, preterm premature rupture of membranes, and a total perinatal mortality rate of 30.7% (4/13). An additional 3 women, who were diagnosed owing to complications outside of pregnancy and at older ages, experienced TTP during their pregnancies, which occurred before diagnosis. Subsequent pregnancies were treated with fresh frozen plasma leading to a 100% fetal survival rate in the pregnancies that reached fetal viability. All placentas had lesions consistent with maternal vascular underperfusion. However, the severity and frequency of these lesions were lower in the 8 placentas from pregnancies treated with fresh frozen plasma. CONCLUSION: This case series details a distinctive cohort of congenital TTP patients, all homozygous for the same, novel ADAMTS13 variant, who presented with clinical complications during pregnancy and maternal vascular lesions of underperfusion in the placenta. Our findings imply that the variant identified in the ADAMTS13 gene in our cohort may have a specific functional impact on the placenta, and that treatment with fresh frozen plasma during pregnancy ameliorates the course of the disease, leading to a milder phenotype or a normal pregnancy in the majority of cases.

Inflammasome components and ADAMTS4 in premature rupture of membranes.

Inflammation may be responsible for the development of premature rupture of membranes (PROM) including preterm PROM (PPROM) and mature PROM (MPROM). A total of four classic receptor proteins have been confirmed to assemble inflammasomes: NLR family pyrin domain containing (NLRP)1, NLRP3 and NLR family CARD­domain containing 4 (NLRC4) and absent in melanoma 2 (AIM2). The activation and expression of these receptor­modulated inflammasomes in placenta and fetal membrane of PROM pregnancies requires investigation. In addition, a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) is a risk factor for PROM, but whether its expression is associated with inflammasome activation remains to be elucidated. In the present study, the placenta and fetal membrane tissues of patients who had suffered PPROM and MPROM and healthy pregnancies were investigated. Reverse transcription­quantitative PCR was used to determine the mRNA expression of inflammasomes and ADAMTS4. Western blotting, immunohistochemistry and ELISA were used to investigate the protein expression levels of inflammasomes and ADAMTS4. The results demonstrated that all four inflammasomes were elevated in placenta and fetal membrane of PPROMs as were mRNA and protein expression levels of IL­18 and IL­1ß (compared with controls). A further increase of inflammasomes and interleukins was observed in MPROMs compared with controls. Similar results were also observed in ADAMTS4 expression in PPROM and MPROM groups. However, immunohistochemistry results revealed no significant difference of inflammasome receptor expression in PPROMs compared with controls. Finally, a general positive correlation between ADAMTS4 and all four inflammasome receptors in placenta and fetal membrane of PPROMs and MPROMs was observed. The present study revealed that NLRP1, NLRP3, AIM2 and NLRC4 inflammasome activation in PROM was increased. Promoted ADAMTS4 level was further observed in PROM group and was significantly correlated with inflammasome expression. Inhibition of inflammasome activation may provide a therapeutic target for clinical PROM treatment.

Inflammation-related downregulation of zonula Occludens-1 in fetal membrane contributes to development of prelabor rupture of membranes.

INTRODUCTION: The aim of this research was to study the alteration of three key tight junction proteins, to explore whether they were involved in the occurrence of prelabor rupture of the membrane (PROM) and to determine the correlation with intrauterine infection. METHODS: A total of 208 women were enrolled between January 2015 to December 2018, including those with preterm and term PROM (PROM group) and normal pregnancies with intact fetal membrane (control group). We investigated the expressions of three key TJ molecules (Zonula occludens-1, Occludin and Claudin-5) in fetal membranes. The localization and expression of Zonula occludens-1 (ZO-1) in the amnion and chorion were studied by immunohistochemistry assay. The associations between ZO-1 expression levels and extent of inflammatory reactions as well as other obstetric characteristics were further studied using Spearman's rank correlation test and Mann-Whitney U test. RESULTS: ZO-1 was significantly downregulated in PROM group compared with control group (P < 0.001), whereas no significant changes were found for Occludin and Claudin-5. ZO-1 expression was reduced in the chorion and amnion layers in PROM group compared with that in control group, which showed a significant difference (P < 0.01), but no significant differences were observed between the preterm PROM and term PROM groups (P > 0.05). The expression levels of ZO-1 in the chorion were negatively correlated with the stage/grade of acute chorioamnionitis (P < 0.05). DISCUSSION: Our study suggests that inflammation-related downregulation of ZO-1 might be a pivotal event in the occurrence of PROM, which helps to clarify the mechanism of membrane rupture caused by infection.

Regulation of Keap-1/Nrf2 Signaling Pathway Is Activated by Oxidative Stress in Patients with Premature Rupture of Membranes.

BACKGROUND The potential mechanisms underlying premature rupture of membrane (PROM) is still unknown. The aim of this study was to determine the role of Keap-1/Nrf2 signaling pathway activation by oxidative stress in patients with preterm premature rupture of membranes. MATERIAL AND METHODS Placental tissues from preterm premature rupture of membranes (PPROM) (n=20), full-term premature rupture of membranes (FPROM) (n=20), and normal-term births (n=20) were collected and amniotic tissues were separated from the placental tissues from pregnant women at Shandong Provincial Qianfoshan Hospital. RT-PCR and Western blot were used to detect the levels of factors in the Keap-1/Nrf2 signaling pathway. To investigate the roles of Nrf2, we downregulated Nrf2 expression using siRNA in primary human amniotic epithelial (HAE) cells. RESULTS Among the control group, FPROM group, and PPROM group, the reactive oxygen species (ROS) levels were significantly increased in the FPROM and PPROM groups. The differences indicated higher levels of oxidative stress in amniotic tissues with FPROM and PPROM after downregulation of si-Nrf2 in HAE cells. Antioxidants were lower in amniotic tissues with the FPROM group and PPROM group than in the control group. The antioxidant enzymes catalase (CAT), glutathione (GSH), glutathione peroxidase (GSHPx), and superoxide dismutases (SOD1 and SOD2) were examined in amniotic tissues. We found that the ROS levels were significantly increased after downregulation of si-Nrf2 compared with the control group. We found that the expression of Heme Oxygenase-1 (HO-1) and Glycogen Synthase Kinase-3ß (GSK-3ß), which is critical in the Keap-1/Nrf2 signaling pathway, increased significantly after downregulation of si-Nrf2 in HAE cells. CONCLUSIONS We found that increased ROS levels and decreased antioxidant enzymes in the PPROM and FPROM patients compared with the control group.

Preterm premature rupture of membranes: prediction of risks in women of Zaporizhzhia region of Ukraine.

The etiology of preterm premature rupture of membranes (PPROM), which is responsible for approximately 30% cases of preterm birth (PTB) is not yet fully understood. AIM: The aim of the study was to create a mathematical model for prognostication of PPROM based on the anamnesis, clinical data, laboratory findings and genetics predictors. MATERIALS AND METHODS: The study involved 80 women with PPROM (between 26 and 34 weeks of gestation) and 50 women having term birth (>37 weeks of gestation) of Zaporizhzhia region of Ukraine. Anamnesis, clinical, laboratory data and single nucleotide polymorphism sequencing of interleukin1 ß (IL1ß), tumor necrosis factor α(TNFα), interleukin4 (IL4), interleukin10 (IL10) and Relaxin 2 (RLN2) genes has been analyzed. Receiver operating characteristic analysis and multivariate logistic regression were used to PPROM predictors identification. RESULTS: We have identified prognostic anamnestic (history of preterm birth), clinical (cervical insuffiency, compromised uteroplacental and fetal circulation), microbiological (vaginal dysbiosis) and hematological criteria for intra-amniotic contamination and further development of PPROM and PTB: WBC>12.3×109/L, GRAN>76%, LYM<19%, neutrophil lymphocyte ratio>3.87, Kalph-Kaliph leukocyte index of intoxication (LII) >3.4, Ostrovsky LII >2.8. Also we have found that GG genotype of IL10 gene polymorphism (rs1800872) leads to a 12.5-fold and CT genotype of RLN2 gene polymorphism (rs4742076) leads to a 17.0-fold increase in risk for PPROM. CONCLUSIONS: The prognostic model that we have suggested is an adequate and convenient instrument for practical medical use, which allows for assessment of PPROM probability with a 85% sensitivity and a 72% specificity.

Antibody Microarray Analysis of Plasma Proteins for the Prediction of Histologic Chorioamnionitis in Women With Preterm Premature Rupture of Membranes.

We aimed to identify maternal blood biomarkers predictive of histologic chorioamnionitis (HCA) in the plasma of women with preterm premature rupture of membranes (PPROM) and to determine whether the combination of these biomarkers with conventional clinical variables can improve the prediction of HCA. This retrospective cohort study included 82 consecutive women with PPROM (23-34 gestational weeks) who delivered within 96 hours of blood sampling. A membrane-based human antibody microarray was used to analyze the plasma proteome. The validation of 5 candidate biomarkers of interest was performed by enzyme-linked immunosorbent assay (ELISA) in the final cohort (n = 82). Serum C-reactive protein (CRP) levels were measured at sampling. Seventy-nine molecules studied exhibited intergroup differences. Validation by ELISA confirmed higher levels of plasma matrix metalloproteinase-9 (MMP-9), interleukin-6 (IL-6), S100 A8/A9, and insulin-like growth factor-binding protein 1 (IGFBP-1), but not tissue inhibitor of metalloproteinase 1 (TIMP-1), in women with HCA than in women without HCA. Using a stepwise regression analysis, a combined prediction model was developed, which included the plasma MMP-9, serum CRP levels, and gestational age (area under the curve [AUC], 0.932). The AUC for this model was significantly greater than that for any single variable included in the predictive model. Protein-antibody microarray technology can be useful in identifying plasma-based predictors for HCA. This study suggests that plasma MMP-9, IL-6, IGFBP-1, and S100 A8/A9 are important noninvasive predictors for HCA in women with PPROM and that the best predictive model, which combined these biomarkers with conventional clinical factors, can significantly improve the predictability for HCA.

Discovery of rare ancestry-specific variants in the fetal genome that confer risk of preterm premature rupture of membranes (PPROM) and preterm birth.

BACKGROUND: Preterm premature rupture of membranes (PPROM) is the leading identifiable cause of preterm birth, a complication that is more common in African Americans. Attempts to identify genetic loci associated with preterm birth using genome-wide association studies (GWAS) have only been successful with large numbers of cases and controls, and there has yet to be a convincing genetic association to explain racial/ethnic disparities. Indeed, the search for ancestry-specific variants associated with preterm birth has led to the conclusion that spontaneous preterm birth could be the consequence of multiple rare variants. The hypothesis that preterm birth is due to rare genetic variants that would go undetected in standard GWAS has been explored in the present study. The detection and validation of these rare variants present challenges because of the low allele frequency. However, some success in the identification of fetal loci/genes associated with preterm birth using whole genome sequencing and whole exome sequencing (WES) has recently been reported. While encouraging, this is currently an expensive technology, and methods to leverage the sequencing data to quickly identify and cost-effectively validate variants are needed. METHODS: We developed a WES data analysis strategy based on neonatal genomic DNA from PPROM cases and term controls that was unencumbered by preselection of candidate genes, and capable of identifying variants in African Americans worthy of focused evaluation to establish statistically significant associations. RESULTS: We describe this approach and the identification of damaging nonsense variants of African ancestry in the DEFB1 and MBL2 genes that encode anti-microbial proteins that presumably defend the fetal membranes from infectious agents. Our approach also enabled us to rule out a likely contribution of a predicted damaging nonsense variant in the METTL7B gene. CONCLUSIONS: Our findings support the notion that multiple rare population-specific variants in the fetal genome contribute to preterm birth associated with PPROM.

Maternal human telomerase reverse transcriptase variants are associated with preterm labor and preterm premature rupture of membranes.

OBJECTIVE: Premature aging and short telomere lengths of fetal tissues are associated with spontaneous preterm labor (PTL) and preterm premature rupture of membranes (pPROM). Maintenance of telomere length is performed by the enzyme telomerase. Human telomerase reverse transcriptase (hTERT) is a subunit of telomerase, and its dysfunction affects telomere shortening. This study assessed whether maternal or fetal genetic variations in the hTERT gene are associated with PTL or pPROM. METHODS: A case (PTL or pPROM) control (term birth) genetic association study was conducted in 654 non-Hispanic white mothers (438 term, 162 PTL, 54 pPROM) and 502 non-Hispanic white newborns (346 term, 116 PTB, 40 pPROM). Maternal and fetal DNA samples were genotyped for 23 single nucleotide polymorphisms (SNPs) within the hTERT gene. Allele frequencies were compared between cases and controls, stratified by PTL and pPROM. Maternal and fetal data were analyzed separately. RESULTS: Allelic differences in one SNP of hTERT (rs2853690) were significantly associated with both PTL (adjusted OR 2.24, 95%CI 1.64-3.06, p = 2.32e-05) and with pPROM (adjusted OR 7.54, 95%CI 3.96-14.33, p = 2.39e-07) in maternal DNA. There was no significant association between the hTERT SNPs analyzed and PTL or pPROM in the fetal samples. CONCLUSION: hTERT polymorphisms in fetal DNA do not associate with PTL or pPROM risk; however, maternal genetic variations in hTERT may play a contributory role in risk of PTL and PPROM.

Infection-induced thrombin production: a potential novel mechanism for preterm premature rupture of membranes (PPROM).

BACKGROUND: Preterm premature rupture of membranes is a leading contributor to maternal and neonatal morbidity and death. Epidemiologic and experimental studies have demonstrated that thrombin causes fetal membrane weakening and subsequently preterm premature rupture of membranes. Although blood is suspected to be the likely source of thrombin in fetal membranes and amniotic fluid of patients with preterm premature rupture of membranes, this has not been proved. Ureaplasma parvum is emerging as a pathogen involved in prematurity, which includes preterm premature rupture of membranes; however, until now, prothrombin production that has been induced directly by bacteria in fetal membranes has not been described. OBJECTIVE: This study was designed to investigate whether Ureaplasma parvum exposure can induce prothrombin production in fetal membranes cells. STUDY DESIGN: Primary fetal membrane cells (amnion epithelial, chorion trophoblast, and decidua stromal) or full-thickness fetal membrane tissue explants from elective, term, uncomplicated cesarean deliveries were harvested. Cells or tissue explants were infected with live Ureaplasma parvum (1×105, 1×106 or 1×107 colony-forming units per milliliter) or lipopolysaccharide (Escherichia coli J5, L-5014; Sigma Chemical Company, St. Louis, MO; 100 ng/mL or 1000 ng/mL) for 24 hours. Tissue explants were fixed for immunohistochemistry staining of thrombin/prothrombin. Fetal membrane cells were fixed for confocal immunofluorescent staining of the biomarkers of fetal membrane cell types and thrombin/prothrombin. Protein and messenger RNA were harvested from the cells and tissue explants for Western blot or quantitative reverse transcription polymerase chain reaction to quantify thrombin/prothrombin protein or messenger RNA production, respectively. Data are presented as mean values ± standard errors of mean. Data were analyzed using 1-way analysis of variance with post hoc Dunnett's test. RESULTS: Prothrombin production and localization were confirmed by Western blot and immunostainings in all primary fetal membrane cells and tissue explants. Immunofluorescence observations revealed a perinuclear localization of prothrombin in amnion epithelial cells. Localization of prothrombin in chorion and decidua cells was perinuclear and cytoplasmic. Prothrombin messenger RNA and protein expression in fetal membranes were increased significantly by Ureaplasma parvum, but not lipopolysaccharide, treatments in a dose-dependent manner. Specifically, Ureaplasma parvum at a dose of 1×107 colony-forming units/mL significantly increased both prothrombin messenger RNA (fold changes in amnion: 4.1±1.9; chorion: 5.7±4.2; decidua: 10.0±5.4; fetal membrane: 9.2±3.0) and protein expression (fold changes in amnion: 138.0±44.0; chorion: 139.6±15.1; decidua: 56.9±29.1; fetal membrane: 133.1±40.0) compared with untreated control subjects. Ureaplasma parvum at a dose of 1×106 colony-forming units/mL significantly up-regulated prothrombin protein expression in chorion cells (fold change: 54.9±5.3) and prothrombin messenger RNA expression in decidua cells (fold change: 4.4±1.9). CONCLUSION: Our results demonstrate that prothrombin can be produced directly by fetal membrane amnion, chorion, and decidua cells. Further, prothrombin production can be stimulated by Ureaplasma parvum exposure in fetal membranes. These findings represent a potential novel underlying mechanism of Ureaplasma parvum-induced rupture of fetal membranes.