Extracellular vesicles (EVs) are nano-scaled vesicles released from all cell types into extracellular fluids and specifically contain signature molecules of the original cells and tissues, including the placenta. Placenta-derived EVs can be detected in maternal circulation at as early as six weeks of gestation, and their release can be triggered by the oxygen level and glucose concentration. Placental-associated complications such as preeclampsia, fetal growth restriction, and gestational diabetes have alterations in placenta-derived EVs in maternal plasma, and this can be used as a liquid biopsy for the diagnosis, prediction, and monitoring of such pregnancy complications. Alpha-thalassemia major (“homozygous alpha-thalassemia-1”) or hemoglobin Bart’s disease is the most severe form of thalassemia disease, and this condition is lethal for the fetus. Women with Bart’s hydrops fetalis demonstrate signs of placental hypoxia and placentomegaly, thereby placenta-derived EVs provide an opportunity for a non-invasive liquid biopsy of this lethal condition. In this article, we introduced clinical features and current diagnostic markers of Bart’s hydrops fetalis, extensively summarize the characteristics and biology of placenta-derived EVs, and discuss the challenges and opportunities of placenta-derived EVs as part of diagnostic tests for placental complications focusing on Bart’s hydrop fetalis.

Background: Intra-amniotic infection has a strong causal association with spontaneous preterm birth and preterm prelabor rupture of membranes (PPROM). The most common route of intra-amniotic infection is the ascending pathway in which microorganisms from the vagina gain access to the amniotic cavity. Distant microorganisms such as those from the oral cavity have been reported in intra-amniotic infection through hematogenous spreading.Case presentation: A 31-year-old gravida 1, para 0 Thai woman at 33⁺⁶ weeks’ gestation presented with leakage of vaginal fluid and irregular uterine contraction. She developed fever at 4 h after admission and was later diagnosed with acute chorioamnionitis. A Cesarean section was performed to terminate pregnancy. In addition to a blood culture, the cultures of amniotic fluid, vaginal and chorioamniotic membrane swabs were positive forStreptococcus mitiswith identical susceptibility profiles. After the delivery and antibiotic prescription, oral examination showed dental caries and chronic periodontitis.Conclusions: This is the first case report demonstrating maternal septicemia and intra-amniotic infection caused byS. mitiswhich might be attributed to periodontitis in women presenting with preterm PROM. We highlighted the association of periodontal disease and preterm labor/PROM syndrome. Oral cavity examination should be included in the prenatal care to ensure good dental hygiene.

Objectives: Early diagnosis and treatment of intra-amniotic infection is crucial. Rapid pathogen identification allows for a definite diagnosis and enables proper management. We determined whether the 16S amplicon sequencing performed by a nanopore sequencing technique make possible rapid bacterial identification at the species level in intra-amniotic infection. Methods: Five cases of confirmed intra-amniotic infection, determined by either cultivation or 16S rDNA polymerase chain reaction (PCR) Sanger sequencing, and 10 cases of women who underwent mid-trimester genetic amniocentesis were included. DNA was extracted from amniotic fluid and PCR was performed on the full-length 16S rDNA. Nanopore sequencing was performed. The results derived from nanopore sequencing were compared with those derived from cultivation and Sanger sequencing methods. Results: Bacteria were successfully detected from amniotic fluid using nanopore sequencing in all cases of intra-amniotic infection. Nanopore sequencing identified additional bacterial species and polymicrobial infections. All patients who underwent a mid-trimester amniocentesis had negative cultures, negative 16S PCR Sanger sequencing and nanopore sequencing. Identification of the microorganisms using nanopore sequencing technique at the bacterial species level was achieved within 5–9 h from DNA extraction. Conclusions: This is the first study demonstrating that the nanopore sequencing technique is capable of rapid diagnosis of intra-amniotic infection using fresh amniotic fluid samples.

Objectives: Streptococcus agalactiae, or Group B Streptococcus (GBS), is a leading cause of neonatal sepsis. Materno-fetal transmission of the microorganisms present in the lower genital tract/perineum is considered to be the most frequent mode for acquisition of infection. It has also been proposed that, in a subset of cases, GBS causes acute chorioamnionitis, intraamniotic infection, and fetal/neonatal sepsis. However, the evidence to support this ascending pathway is derived from microbiologic studies that rely on cultivation methods, which do not have the resolution to determine if the microorganisms causing neonatal sepsis are the same as those found in the amniotic fluid and the vaginal ecosystem. Methods: We used whole genome sequencing of the microorganisms isolated from the vagina, amniotic fluid, chorioamniotic membranes, and neonatal blood (four isolates) in a case of early neonatal sepsis. Using hybrid genome assembly, we characterized the genomic features including virulence factors and antimicrobial resistance in four isolates from the same mother, placenta, and newborn. Results: Whole genome sequencing revealed that the microorganisms in the four clinical isolates corresponded to S. agalactiae sequence type 1, clonal complexes 1, and serotype Ib. Comparative genomic analysis illustrated similar DNA sequences of the four genomes. Conclusions: This study presents the first evidence of the genomic similarity of microorganisms in the vaginal ecosystem, the space between the chorioamniotic membranes of the placenta, amniotic fluid, and neonatal blood.

Intraamniotic infection (defined as intraamniotic inflammation with microorganisms) is an important cause of the preterm labor syndrome. Methods for the detection of microorganisms in amniotic fluid are culture and/or polymerase chain reaction assay. However, both methods take time, and the results are rarely available for clinical decision-making. Nanopore sequencing technology offers real-time, long-read sequencing that can produce rapid results. To determine 1) the diagnostic performance of the 16S rDNA nanopore sequencing method for the identification of microorganisms in patients with intraamniotic inflammation and 2) the relationship between microbial burden and the intensity of the amniotic fluid inflammatory response. We performed a prospective cohort study that included singleton pregnancies presenting with symptoms of preterm labor with intact membranes or of preterm prelabor rupture of the membranes. Amniotic fluid samples were obtained for the evaluation of bacteria in the amniotic cavity using cultivation and polymerase chain reaction-based 16S Sanger sequencing methods. Participants were classified into 4 groups according to the results of an amniotic fluid culture, 16S Sanger sequencing, and an amniotic fluid interleukin 6 concentration: 1) no intraamniotic infection and intraamniotic inflammation (interleukin 6 <2.6 ng/mL, and no microorganisms in the amniotic cavity, as determined by culture or 16S Sanger sequencing); 2) microbial invasion of the amniotic cavity without intraamniotic inflammation, defined by the presence of bacteria detected by culture or 16S Sanger sequencing; 3) sterile intraamniotic inflammation (interleukin 6 ≥2.6 ng/mL without microbial invasion of the amniotic cavity); and 4) intraamniotic infection (interkeukin 6 ≥2.6 ng/mL with microbial invasion of the amniotic cavity). Patients who underwent a mid-trimester amniocentesis, had no intraamniotic infection or intraamniotic inflammation, and delivered at term represented the control group. 16S rDNA nanopore sequencing was performed and the diagnostic indices for the identification of intraamniotic infection were determined. Bioinformatic analysis was carried out to identify microorganisms, and a read count of at least 100 or a read count exceeding that of the background species from the control group, along with a relative abundance of no less than 1%, was used. 1) The 16S nanopore sequencing had a sensitivity of 88.9% (8/9), specificity of 95.4% (41/43), positive predictive value of 80.0% (8/10), negative predictive value of 97.6% (41/42), positive likelihood ratio of 19.1 (95% confidence interval, 4.8-75.4), negative likelihood ratio of 0.1 (95% confidence interval, 0.02-0.7), and an accuracy of 94.2% (49/52) for the identification of intraamniotic infection (prevalence, 17% [9/52]); 2) the microbial load determined by the 16S nanopore sequencing had a strong positive correlation with the intensity of an intraamniotic inflammatory response (amniotic fluid interleukin 6 concentration; Spearman's correlation 0.9; P=.002); and 3) a subgroup of patients with intraamniotic inflammation did not have bacteria determined by culture, Sanger sequencing, or nanopore 16S, thus confirming the existence of sterile intraamniotic inflammation. The 16S nanopore sequencing has high diagnostic indices, predictive values, likelihood ratios, and accuracy in the diagnosis of intraamniotic infection.

Background: Preterm labor syndrome is associated with high perinatal morbidity and mortality, and intra-amniotic infection is a cause of preterm labor. The standard identification of causative microorganisms is based on the use of biochemical phenotypes, together with broth dilution-based antibiotic susceptibility from organisms grown in culture. However, such methods could not provide an accurate epidemiological aspect and a genetic basis of antimicrobial resistance leading to an inappropriate antibiotic administration. Hybrid genome assembly is a combination of short- and long-read sequencing, which provides better genomic resolution and completeness for genotypic identification and characterization. Herein, we performed a hybrid whole genome assembly sequencing of a pathogen associated with acute histologic chorioamnionitis in women presenting with PPROM. Results: We identified Enterococcus faecium, namely E. faecium strain RAOG174, with several antibiotic resistance genes, including vancomycin and aminoglycoside. Virulence-associated genes and potential bacteriophage were also identified in this genome. Conclusion: We report herein the first study demonstrating the use of hybrid genome assembly and genomic analysis to identify E. faecium ST17 as a pathogen associated with acute histologic chorioamnionitis. The analysis provided several antibiotic resistance-associated genes/mutations and mobile genetic elements. The occurrence of E. faecium ST17 raised the awareness of the colonization of clinically relevant E. faecium and the carrying of antibiotic resistance. This finding has brought the advantages of genomic approach in the identification of the bacterial species and antibiotic resistance gene for E. faecium for appropriate antibiotic use to improve maternal and neonatal care.